Application of High-Resolution UPLC-MSE/TOF Confirmation in Forensic Urine Drug Screening by UPLC-MS/MS.

The transition from presumptive (immunoassay) drug screening to definitive screening has continued in the practice of analytical toxicology. Development of a ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) screening method for over sixty drugs and metabolites (analytes) in urine has been reported by the authors and has been applied in probation, drug court, social services, chemical dependency, pain management and addiction medicine casework. Testing by the definitive screening method has increased both the rate and diversity of initial-positive drug findings, due to the lower positive thresholds and wider panel of analytes. Use of definitive screening in forensic casework, however, requires retesting of initial-positive analytes using a second method based upon a different analytical technique with at least equivalent sensitivity and selectivity. Consequently, a UPLC-MSE/TOF method for confirmation of the initial-positive analytes has been adapted; the method is for targeted confirmation and is based upon an alternate mass spectrometry technology and column separation. Both the initial screen and the confirmatory analysis employ threshold accurate calibration for normalization of matrix effects, without the use of stable isotopes. Validation and application of the complete workflow, in forensic urine drug testing casework, is reported.

Screening with Quantification for 64 Drugs and Metabolites in Human Urine using UPLC-MS-MS Analysis and a Threshold Accurate Calibration.

Drug and metabolite (analytes) identification together with quantification is an important analytical tool in forensic and clinical toxicology. We report the development and validation of a definitive detection and quantification method (UPLC-MS-MS) for initial screening of 64 analytes in urine. The principle of the method is a quantitative extension of a recently reported threshold accurate calibration (TAC) technique which employs a rapid dual-specimen analysis i.e., with and without addition of a reference-analyte standard for normalization of matrix effects. Analytes include pharmaceutical and illicit agents from opiate and opioid agonist, opiate-antagonist, benzodiazepine, amphetamine, designer amphetamine, cathinone, cocaine, hallucinogen, gabapentinoid and sedative drug classes. Using a 96-well plate format, the protocol employs glucuronidase hydrolysis, 27-fold urine dilution and a 3 min UPLC-MS-MS acquisition. Subsequent data management includes calculation of a normalized TAC ratio response and weighted least squares calibration. The method utilizes analyte-specific calibration ranges from 2.5 to 1,500 ng/mL with quality control (QC) monitoring of transition-ion ratio, calibrator re-analysis, injection precision and multi-level QC analysis. Method precision, bias, calibration linearity, detection limit, carryover, crossover studies and external proficiency performance were evaluated based on pre-established criteria. The validated method provides an alternative to stable-isotope internal standardization methods of quantification and is applicable to screening with quantification in routine toxicology practice.

Definitive Drug and Metabolite Screening in Urine by UPLC-MS-MS Using a Novel Calibration Technique.

Drug screening is an essential analytical tool for detection of therapeutic, illicit and emerging drug use. Presumptive immunoassay screening is widely used, while initial definitive testing by chromatography-coupled mass spectrometry is hampered due to complex pre-analysis steps, long chromatography time and matrix effects. The aim of this study is to develop and validate a definitive test for rapid and threshold accurate screening of 33 drugs or metabolites (analytes) in urine. Sample preparation in a 96-well plate format involves rapid glucuronidase hydrolysis followed by dilution, filtration and ultra-performance liquid chromatography-MS-MS analysis. Chromatographic separation, on an ACQUITY UPLC® BEH phenyl column is optimized for a 3-min MS-MS ion acquisition. Matrix effect was normalized by an innovative technique called threshold accurate calibration employing an additional analysis with an analyte spike as an internal standard undergoing the same matrix effect as an analyte in a drug-positive donor specimen. Accuracy and precision, at above and below threshold concentrations, were determined by replicate analysis of control urine pools containing 50, 75, 125 and 150% of threshold concentrations. Accuracy and selectivity were further demonstrated by concordant findings in proficiency and confirmatory testing. The study shows the applicability of definitive testing as an alternative to immunoassay screening and demonstrates a new approach to normalization of matrix effect.

Identification of novel genes that regulate androgen receptor signaling and growth of androgen-deprived prostate cancer cells.

Prostate cancer progression to castration refractory disease is associated with anomalous transcriptional activity of the androgen receptor (AR) in an androgen-depleted milieu. To identify novel gene products whose downregulation transactivates AR in prostate cancer cells, we performed a screen of enzymatically-generated shRNA lenti-libraries selecting for transduced LNCaP cells with elevated expression of a fluorescent reporter gene under the control of an AR-responsive promoter. The shRNAs present in selected populations were analyzed using high-throughput sequencing to identify target genes. Highly enriched gene targets were then validated with siRNAs against selected genes, testing first for increased expression of luciferase from an AR-responsive promoter and then for altered expression of endogenous androgen-regulated genes in LNCaP cells. We identified 20 human genes whose silencing affected the expression of exogenous and endogenous androgen-responsive genes in prostate cancer cells grown in androgen-depleted medium. Knockdown of four of these genes upregulated the expression of endogenous AR targets and siRNAs targeting two of these genes (IGSF8 and RTN1) enabled androgen-independent proliferation of androgen-dependent cells. The effects of IGSF8 appear to be mediated through its interaction with a tetraspanin protein, CD9, previously implicated in prostate cancer progression. Remarkably, homozygous deletions of IGSF8 are found almost exclusively in prostate cancers but not in other cancer types. Our study shows that androgen independence can be achieved through the inhibition of specific genes and reveals a novel set of genes that regulate AR signaling in prostate cancers.

Interactome analysis reveals ezrin can adopt multiple conformational states.

Ezrin, a member of the ezrin-radixin-moesin family (ERM), is an essential regulator of the structure of microvilli on the apical aspect of epithelial cells. Ezrin provides a linkage between membrane-associated proteins and F-actin, oscillating between active/open and inactive/closed states, and is regulated in part by phosphorylation of a C-terminal threonine. In the open state, ezrin can bind a number of ligands, but in the closed state the ligand-binding sites are inaccessible. In vitro analysis has proposed that there may be a third hyperactivated form of ezrin. To gain a better understanding of ezrin, we conducted an unbiased proteomic analysis of ezrin-binding proteins in an epithelial cell line, Jeg-3. We refined our list of interactors by comparing the interactomes using quantitative mass spectrometry between wild-type ezrin, closed ezrin, open ezrin, and hyperactivated ezrin. The analysis reveals several novel interactors confirmed by their localization to microvilli, as well as a significant class of proteins that bind closed ezrin. Taken together, the data indicate that ezrin can exist in three different conformational states, and different ligands "perceive" ezrin conformational states differently.

DNA-repair scaffolds dampen checkpoint signalling by counteracting the adaptor Rad9.

In response to genotoxic stress, a transient arrest in cell-cycle progression enforced by the DNA-damage checkpoint (DDC) signalling pathway positively contributes to genome maintenance. Because hyperactivated DDC signalling can lead to a persistent and detrimental cell-cycle arrest, cells must tightly regulate the activity of the kinases involved in this pathway. Despite their importance, the mechanisms for monitoring and modulating DDC signalling are not fully understood. Here we show that the DNA-repair scaffolding proteins Slx4 and Rtt107 prevent the aberrant hyperactivation of DDC signalling by lesions that are generated during DNA replication in Saccharomyces cerevisiae. On replication stress, cells lacking Slx4 or Rtt107 show hyperactivation of the downstream DDC kinase Rad53, whereas activation of the upstream DDC kinase Mec1 remains normal. An Slx4-Rtt107 complex counteracts the checkpoint adaptor Rad9 by physically interacting with Dpb11 and phosphorylated histone H2A, two positive regulators of Rad9-dependent Rad53 activation. A decrease in DDC signalling results from hypomorphic mutations in RAD53 and H2A and rescues the hypersensitivity to replication stress of cells lacking Slx4 or Rtt107. We propose that the Slx4-Rtt107 complex modulates Rad53 activation by a competition-based mechanism that balances the engagement of Rad9 at replication-induced lesions. Our findings show that DDC signalling is monitored and modulated through the direct action of DNA-repair factors.

Local phosphocycling mediated by LOK/SLK restricts ezrin function to the apical aspect of epithelial cells.

In this paper, we describe how a dynamic regulatory process is necessary to restrict microvilli to the apical aspect of polarized epithelial cells. We found that local phosphocycling regulation of ezrin, a critical plasma membrane-cytoskeletal linker of microvilli, was required to restrict its function to the apical membrane. Proteomic approaches and ribonucleic acid interference knockdown identified lymphocyte-oriented kinase (LOK) and SLK as the relevant kinases. Using drug-resistant LOK and SLK variants showed that these kinases were sufficient to restrict ezrin function to the apical domain. Both kinases were enriched in microvilli and locally activated there. Unregulated kinase activity caused ezrin mislocalization toward the basolateral domain, whereas expression of the kinase regulatory regions of LOK or SLK resulted in local inhibition of ezrin phosphorylation by the endogenous kinases. Thus, the domain-specific presence of microvilli is a dynamic process requiring a localized kinase driving the phosphocycling of ezrin to continually bias its function to the apical membrane.

DNA damage signaling recruits the Rtt107-Slx4 scaffolds via Dpb11 to mediate replication stress response.

The DNA damage checkpoint kinase Mec1(ATR) is critical for maintaining the integrity of replication forks. Though it has been proposed to promote fork repair, the mechanisms by which Mec1 regulates DNA repair factors remain unclear. Here, we found that Mec1 mediates a key interaction between the fork protein Dpb11 and the DNA repair scaffolds Slx4-Rtt107 to regulate replication stress response. Dissection of the molecular basis of the interaction reveals that Slx4 and Rtt107 jointly bind Dpb11 and that Slx4 phosphorylation is required. Mutation of Mec1 phosphorylation sites in Slx4 disrupts its interaction with Dpb11 and compromises the cellular response to replisomes blocked by DNA alkylation. Multiple fork repair factors associate with Rtt107 or Slx4, supporting that Mec1-dependent assembly of the Rtt107-Slx4-Dpb11 complex functions to coordinate fork repair. Our results unveil how Mec1 regulates the Slx4 and Rtt107 scaffolds and establish a mechanistic link between DNA damage signaling and fork repair.

Androgenic regulation of hedgehog signaling pathway components in prostate cancer cells.

Hedgehog signaling is thought to play a role in several human cancers including prostate cancer. Although prostate cancer cells express many of the gene products involved in hedgehog signaling, these cells are refractory to the canonical signaling effects of exogenous hedgehog ligands or to activated Smoothened, the hedgehog-regulated mediator of Gli transcriptional activation. Here, we show that the expression of hedgehog ligands and some hedgehog target genes are regulated by androgen in the human prostate cancer cell line, LNCaP and its more metastatic variants (C4-2 and C4-2B). Androgen (R1881) strongly suppressed the expression of hedgehog ligands in these cells and their prolonged maintenance in androgen-deficient medium upregulated Sonic and Indian hedgehog mRNA and protein levels by up to 30,000-fold. Hedgehogs were released into the conditioned medium of androgen-deprived LNCaP cells and this medium was able to increase hedgehog target gene expression in hedgehog-responsive mouse fibroblasts (MC3T3-E1). Moreover, this activity was accompanied by increased expression of Gli target genes, Patched 1 and Gli2, in LNCaP that could be suppressed by cyclopamine, indicating that chronic androgen-deprivation also re-awakens the autocrine responsiveness of the cancer cells to hedgehog. In contrast to the suppressive effects of R1881 on hedgehog ligand and Gli2 expression, we found that Gli1 expression in LNCaP cells was induced by R1881. Given the ability of androgen to modulate the expression and release of hedgehog ligands and the activity of the autocrine hedgehog signaling pathway in these prostate cancer cells, our results imply that chronic androgen deprivation therapy (ADT) for prostate cancer might create a hedgehog signaling environment in the region of the tumor that could ultimately impact on the long term effectiveness of this treatment. This consideration supports the idea of clinically testing hedgehog-blocking drugs in conjunction with ADT in patients with advanced prostate cancer.

Cell adhesion molecule L1 disrupts E-cadherin-containing adherens junctions and increases scattering and motility of MCF7 breast carcinoma cells.

The first steps of invasion and metastasis include the dissociation of adherens junctions and the induction of migratory phenotype, through a program that resembles epithelial-mesenchymal transition (EMT). The L1 cell adhesion molecule, which is normally found primarily in the brain, was recently shown to be expressed in different types of cancer and to have tumor-promoting activity. We now find that L1 mediates EMT-like events in MCF7 breast carcinoma cells. MCF7 predominantly expresses the nonneuronal isoform of L1, as do 16 of 17 other cell lines derived from different types of cancer. L1 protein expression in MCF7 cells, which form E-cadherin-containing adherens junctions, is inversely related to cell density. Analysis of MCF7 cells with overexpression or knockdown of nonneuronal L1 isoform revealed that L1 expression leads to the disruption of adherens junctions and increases beta-catenin transcriptional activity. As a result, L1 expression promotes the scattering of epithelial cells from compact colonies. Expression of the full-length L1 protein, but not of its soluble extracellular moiety, increases the motility of the MCF7 epithelial monolayer in a wound-healing assay, in which L1 expression is preferentially observed and required in cells leading the movement of the monolayer. Based on these results, we propose a model for the role of L1 as a trigger of EMT-like events in transformed epithelial cells.