Modulating sensitivity to drug-induced apoptosis: the future for chemotherapy?

Drug resistance is a fundamental problem in the treatment of most common human cancers. Our understanding of the cellular mechanisms underlying death and survival has allowed the development of rational approaches to overcoming drug resistance. The mitogen activated protein kinase family of protein serine/threonine kinases has been implicated in this complex web of signalling, with some members acting to enhance death and other members to prevent it. A recent publication by MacKeigan et al is the first to demonstrate an enhancement of drug-induced cell death by simultaneous blockade of MEK-mediated survival signalling, and offers the potential for targeted adjuvant therapy as a means of overcoming drug resistance

Human Cell Chips: Adapting DNA Microarray Spotting Technology to Cell-Based Imaging Assays

Here we describe human spotted cell chips, a technology for determining cellular state across arrays of cells subjected to chemical or genetic perturbation. Cells are grown and treated under standard tissue culture conditions before being fixed and printed onto replicate glass slides, effectively decoupling the experimental conditions from the assay technique. Each slide is then probed using immunofluorescence or other optical reporter and assayed by automated microscopy. We show potential applications of the cell chip by assaying HeLa and A549 samples for changes in target protein abundance (of the dsRNA-activated protein kinase PKR), subcellular localization (nuclear translocation of NFκB) and activation state (phosphorylation of STAT1 and of the p38 and JNK stress kinases) in response to treatment by several chemical effectors (anisomycin, TNFα, and interferon), and we demonstrate scalability by printing a chip with ∼4,700 discrete samples of HeLa cells. Coupling this technology to high-throughput methods for culturing and treating cell lines could enable researchers to examine the impact of exogenous effectors on the same population of experimentally treated cells across multiple reporter targets potentially representing a variety of molecular systems, thus producing a highly multiplexed dataset with minimized experimental variance and at reduced reagent cost compared to alternative techniques. The ability to prepare and store chips also allows researchers to follow up on observations gleaned from initial screens with maximal repeatability

Tumours and tremors: how PTEN regulation underlies both

Mutations of the tumour suppressor PTEN (phosphatase and tensin homolog deleted on chromosome 10) are seen in many human cancers. However, dysregulation of PTEN may be involved in other disease states such as Parkinson's disease. This minireview describes recent work examining PTEN regulation and its implications for the development of both cancer and neurodegenerative disease

Characterization of a Human Cell Line Stably Over-Expressing the Candidate Oncogene, Dual Specificity Phosphatase 12

Analysis of chromosomal rearrangements within primary tumors has been influential in the identification of novel oncogenes. Identification of the "driver" gene(s) within cancer-derived amplicons is, however, hampered by the fact that most amplicons contain many gene products. Amplification of 1q21-1q23 is strongly associated with liposarcomas and microarray-based comparative genomic hybridization narrowed down the likely candidate oncogenes to two: the activating transcription factor 6 (atf6) and the dual specificity phosphatase 12 (dusp12). While atf6 is an established transcriptional regulator of the unfolded protein response, the potential role of dusp12 in cancer remains uncharacterized.To evaluate the oncogenic potential of dusp12, we established stable cell lines that ectopically over-express dusp12 in isolation and determined whether this cell line acquired properties frequently associated with transformed cells. Here, we demonstrate that cells over-expressing dusp12 display increased cell motility and resistance to apoptosis. Additionally, over-expression of dusp12 promoted increased expression of the c-met proto-oncogene and the collagen and laminin receptor intergrin alpha 1 (itga1) which is implicated in metastasis.Collectively, these results suggest that dusp12 is oncologically relevant and exposes a potential association between dusp12 and established oncogenes that could be therapeutically targeted

Integrated Functional, Gene Expression and Genomic Analysis for the Identification of Cancer Targets

The majority of new drug approvals for cancer are based on existing therapeutic targets. One approach to the identification of novel targets is to perform high-throughput RNA interference (RNAi) cellular viability screens. We describe a novel approach combining RNAi screening in multiple cell lines with gene expression and genomic profiling to identify novel cancer targets. We performed parallel RNAi screens in multiple cancer cell lines to identify genes that are essential for viability in some cell lines but not others, suggesting that these genes constitute key drivers of cellular survival in specific cancer cells. This approach was verified by the identification of PIK3CA, silencing of which was selectively lethal to the MCF7 cell line, which harbours an activating oncogenic PIK3CA mutation. We combined our functional RNAi approach with gene expression and genomic analysis, allowing the identification of several novel kinases, including WEE1, that are essential for viability only in cell lines that have an elevated level of expression of this kinase. Furthermore, we identified a subset of breast tumours that highly express WEE1 suggesting that WEE1 could be a novel therapeutic target in breast cancer. In conclusion, this strategy represents a novel and effective strategy for the identification of functionally important therapeutic targets in cancer

Cooperation of Mtmr8 with PI3K Regulates Actin Filament Modeling and Muscle Development in Zebrafish

It has been shown that mutations in at least four myotubularin family genes (MTM1, MTMR1, 2 and 13) are causative for human neuromuscular disorders. However, the pathway and regulative mechanism remain unknown.Here, we reported a new role for Mtmr8 in neuromuscular development of zebrafish. Firstly, we cloned and characterized zebrafish Mtmr8, and revealed the expression pattern predominantly in the eye field and somites during early somitogenesis. Using morpholino knockdown, then, we observed that loss-of-function of Mtmr8 led to defects in somitogenesis. Subsequently, the possible underlying mechanism and signal pathway were examined. We first checked the Akt phosphorylation, and observed an increase of Akt phosphorylation in the morphant embryos. Furthermore, we studied the PH/G domain function within Mtmr8. Although the PH/G domain deletion by itself did not result in embryonic defect, addition of PI3K inhibitor LY294002 did give a defective phenotype in the PH/G deletion morphants, indicating that the PH/G domain was essential for Mtmr8's function. Moreover, we investigated the cooperation of Mtmr8 with PI3K in actin filament modeling and muscle development, and found that both Mtmr8-MO1 and Mtmr8-MO2+LY294002 led to the disorganization of the actin cytoskeleton. In addition, we revealed a possible participation of Mtmr8 in the Hedgehog pathway, and cell transplantation experiments showed that Mtmr8 worked in a non-cell autonomous manner in actin modeling.The above data indicate that a conserved functional cooperation of Mtmr8 with PI3K regulates actin filament modeling and muscle development in zebrafish, and reveal a possible participation of Mtmr8 in the Hedgehog pathway. Therefore, this work provides a new clue to study the physiological function of MTM family members

Identification and Replication of Loci Involved in Camptothecin-Induced Cytotoxicity Using CEPH Pedigrees

To date, the Centre d'Etude Polymorphism Humain (CEPH) cell line model has only been used as a pharmacogenomic tool to evaluate which genes are responsible for the disparity in response to a single drug. The purpose of this study was demonstrate the model's ability to establish a specific pattern of quantitative trait loci (QTL) related to a shared mechanism for multiple structurally related drugs, the camptothecins, which are Topoisomerase 1 inhibitors. A simultaneous screen of six camptothecin analogues for in vitro sensitivity in the CEPH cell lines resulted in cytotoxicity profiles and orders of potency which were in agreement with the literature. For all camptothecins studied, heritability estimates for cytotoxic response averaged 23.1±2.6%. Nonparametric linkage analysis was used to identify a relationship between genetic markers and response to the camptothecins. Ten QTLs on chromosomes 1, 3, 5, 6, 11, 12, 16 and 20 were identified as shared by all six camptothecin analogues. In a separate validation experiment, nine of the ten QTLs were replicated at the significant and suggestive levels using three additional camptothecin analogues. To further refine this list of QTLs, another validation study was undertaken and seven of the nine QTLs were independently replicated for all nine camptothecin analogues. This is the first study using the CEPH cell lines that demonstrates that a specific pattern of QTLs could be established for a class of drugs which share a mechanism of action. Moreover, it is the first study to report replication of linkage results for drug-induced cytotoxicity using this model. The QTLs, which have been identified as shared by all camptothecins and replicated across multiple datasets, are of considerable interest; they harbor genes related to the shared mechanism of action for the camptothecins, which are responsible for variation in response

Measurement of the Bottom-Strange Meson Mixing Phase in the Full CDF Data Set

We report a measurement of the bottom-strange meson mixing phase \beta_s using the time evolution of B0_s -> J/\psi (->\mu+\mu-) \phi (-> K+ K-) decays in which the quark-flavor content of the bottom-strange meson is identified at production. This measurement uses the full data set of proton-antiproton collisions at sqrt(s)= 1.96 TeV collected by the Collider Detector experiment at the Fermilab Tevatron, corresponding to 9.6 fb-1 of integrated luminosity. We report confidence regions in the two-dimensional space of \beta_s and the B0_s decay-width difference \Delta\Gamma_s, and measure \beta_s in [-\pi/2, -1.51] U [-0.06, 0.30] U [1.26, \pi/2] at the 68% confidence level, in agreement with the standard model expectation. Assuming the standard model value of \beta_s, we also determine \Delta\Gamma_s = 0.068 +- 0.026 (stat) +- 0.009 (syst) ps-1 and the mean B0_s lifetime, \tau_s = 1.528 +- 0.019 (stat) +- 0.009 (syst) ps, which are consistent and competitive with determinations by other experiments.Comment: 8 pages, 2 figures, Phys. Rev. Lett 109, 171802 (2012