Complementary Screen Research Articles

Abstract 4972: High-throughput functional profiling of dedifferentiated liposarcoma cell lines

Abstract We previously profiled several adult soft-tissue sarcoma subtypes using sequencing, copy number analysis, and gene expression arrays. This high-throughput genetic profiling revealed dozens of candidate genes deserving of further functional validation. To prioritize genes, we performed a pooled shRNA screen to identify essential genes in liposarcoma. Dedifferentiated liposarcoma (DDLS) served as a model for this approach as multiple robust cell lines are available. Five DDLS cell lines (DDLS8817, LPS141, FU-DDLS-1, RDD8107, and LP6) were infected with a pool of 54,020 lentiviral shRNAs targeting ∼11,000 genes (median 5 shRNAs per gene) and passaged for 16 doublings. DNA was extracted and hairpin sequences were PCR amplified and hybridized to a custom microarray that interrogates all shRNAs in the pool. shRNAs were ranked according to their differential abundance between early and late passages. Genes corresponding to the most significantly depleted shRNAs are presumed to affect cell proliferation and are candidate oncogenes. We compared the pattern of hairpin depletion in the DDLS cell lines to a panel of over 60 cell lines representing other common cancers screened with the same pooled shRNA library. The 5 DDLS cell lines clustered into 3 distinct groups. Only one gene appeared to be essential in all 3 groups: WWTR1 (TAZ1), a master-regulator of adipocyte differentiation. Twenty-eight genes appeared essential in at least two DDLS groups including MDM2 and ZBTB2, both reported to negatively regulate the tumor suppressor gene TP53. Low-throughput in vitro experiments have confirmed that WWTR1 knockdown using two shRNA clones inhibits LPS141 proliferation by 78 and 75% after 4 days as compared to scramble (p=0.004 and 0.06 respectively). Our complementary genome-scaled functional screen has confirmed a known oncogene in DDLS and revealed additional candidate genes that appear to have a role in proliferation. Further integration of existing datasets may nominate additional genes essential for liposarcoma pathogenesis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4972. doi:10.1158/1538-7445.AM2011-4972

The 19-Amino Acid Insertion in the Tumor-associated Splice Isoform Rac1b Confers Specific Binding to p120 Catenin

The Rac1b splice isoform contains a 19-amino acid insertion not found in Rac1; this insertion leads to decreased GTPase activity and reduced affinity for GDP, resulting in the intracellular predominance of GTP-bound Rac1b. Here, using co-precipitation and proteomic methods, we find that Rac1b does not bind to many common regulators of Rho family GTPases but that it does display enhanced binding to SmgGDS, RACK1, and p120 catenin (p120(ctn)), proteins involved in cell-cell adhesion, motility, and transcriptional regulation. We use molecular modeling and structure analysis approaches to determine that the interaction between Rac1b and p120(ctn) is dependent upon protein regions that are predicted to be unstructured in the absence of molecular complex formation, suggesting that the interaction between these two proteins involves coupled folding and binding. We also find that directed cell movement initiated by Rac1b is dependent upon p120. These results define a distinct binding functionality of Rac1b and provide insight into how the distinct phenotypic program activated by this protein may be implemented through molecular recognition of effectors distinct from those of Rac1.

Species-specific activation of nuclear receptors correlates with the response of liver drug metabolizing enzymes to EMD 392949 in vitro

We previously reported on the species-specific effects on drug metabolizing enzymes (DME), in particular cytochrome P450-dependent monooxygenases (P450s), by the drug development candidate EMD 392949 (EMD) in vitro and in vivo. Induction of P450s occurs via activation of specific transcription factors such as the arylhydrocarbon receptor (AhR) and the nuclear xenobiotic receptors (NXRs). We analyzed whether the reported species-specific P450 induction by EMD could be related to a specific activation of the CYP1A regulator AhR and the CYP3A regulator pregnane X receptor (PXR) in human and rat cell lines. The human HepG2 and rat H4IIE cell lines exhibited inducibility of CYP1A and 3A and expressed functional AhR as well as PXR. CYP3A was induced by EMD in human HepG2 cells exceeding the level induced by rifampicin, but was not induced in rat H4IIE cells. Regulation of P450s was not related to expression levels of their respective transcription factor, but EMD treatment resulted in a significant reporter gene activation in xenobiotic response enhancer module (XREM)-transfected HepG2 but not H4IIE cells indicating activation of human but not rat PXR. In summary, we showed that the P450 inducing properties of EMD were perfectly reflected by its ability to activate AhR or PXR in a species-specific manner. These findings support the tight correlation of species-specific nuclear receptor activation with P450 induction and foster the use of nuclear receptor activation as a complementary screen to identify cytochrome P450 inducers.

3D Pharmacophore Mapping Using 4D QSAR Analysis for the Cytotoxicity of Lamellarins Against Human Hormone-Dependent T47D Breast Cancer Cells

4D quantitative structure-activity relationship (QSAR) and 3D pharmacophore models were built and investigated for cytotoxicity using a training set of 25 lamellarins against human hormone dependent T47D breast cancer cells. Receptor-independent (RI) 4D QSAR models were first constructed from the exploration of eight possible receptor-binding alignments for the entire training set. Since the training set is small (25 compounds), the generality of the 4D QSAR paradigm was then exploited to devise a strategy to maximize the extraction of binding information from the training set and to also permit virtual screening of diverse lamellarin chemistry. 4D QSAR models were sought for only six of the most potent lamellarins of the training set as well as another subset composed of lamellarins with constrained ranges in molecular weight and lipophilicity. This overall modeling strategy has permitted maximizing 3D pharmacophore information from this small set of structurally complex lamellarins that can be used to drive future analog synthesis and the selection of alternate scaffolds. Overall, it was found that the formation of an intermolecular hydrogen bond and the hydrophobic interactions for substituents on the E ring most modulate the cytotoxicity against T47D breast cancer cells. Hydrophobic substitutions on the F-ring can also enhance cytotoxic potency. A complementary high-throughput virtual screen to the 3D pharmacophore models, a 4D fingerprint QSAR model, was constructed using absolute molecular similarity. This 4D fingerprint virtual high-throughput screen permits a larger range of chemistry diversity to be assayed than with the 4D QSAR models. The optimized 4D QSAR 3D pharmacophore model has a leave-one-out cross-correlation value of xv-r2 = 0.947, while the optimized 4D fingerprint virtual screening model has a value of xv-r2 = 0.719. This work reveals that it is possible to develop significant QSAR, 3D pharmacophore, and virtual screening models for a small set of lamellarins showing cytotoxic behavior in breast cancer screens that can guide future drug development based upon lamellarin chemistry.

Mammalian MagT1 and TUSC3 are required for cellular magnesium uptake and vertebrate embryonic development

Magnesium (Mg(2+)) is the second most abundant cation in cells, yet relatively few mechanisms have been identified that regulate cellular levels of this ion. The most clearly identified Mg(2+) transporters are in bacteria and yeast. Here, we use a yeast complementary screen to identify two mammalian genes, MagT1 and TUSC3, as major mechanisms of Mg(2+) influx. MagT1 is universally expressed in all human tissues and its expression level is up-regulated in low extracellular Mg(2+). Knockdown of either MagT1 or TUSC3 protein significantly lowers the total and free intracellular Mg(2+) concentrations in mammalian cell lines. Morpholino knockdown of MagT1 and TUSC3 protein expression in zebrafish embryos results in early developmental arrest; excess Mg(2+) or supplementation with mammalian mRNAs can rescue the effects. We conclude that MagT1 and TUSC3 are indispensable members of the vertebrate plasma membrane Mg(2+) transport system.

Cross-regulatory protein–protein interactions between Hox and Pax transcription factors

Homeotic Hox selector genes encode highly conserved transcriptional regulators involved in the differentiation of multicellular organisms. Ectopic expression of the Antennapedia (ANTP) homeodomain protein in Drosophila imaginal discs induces distinct phenotypes, including an antenna-to-leg transformation and eye reduction. We have proposed that the eye loss phenotype is a consequence of a negative posttranslational control mechanism because of direct protein-protein interactions between ANTP and Eyeless (EY). In the present work, we analyzed the effect of various ANTP homeodomain mutations for their interaction with EY and for head development. Contrasting with the eye loss phenotype, we provide evidence that the antenna-to-leg transformation involves ANTP DNA-binding activity. In a complementary genetic screen performed in yeast, we isolated mutations located in the N terminus of the ANTP homeodomain that inhibit direct interactions with EY without abolishing DNA binding in vitro and in vivo. In a bimolecular fluorescence complementation assay, we detected the ANTP-EY interaction in vivo, these interactions occurring through the paired domain and/or the homeodomain of EY. These results demonstrate that the homeodomain supports multiple molecular regulatory functions in addition to protein-DNA and protein-RNA interactions; it is also involved in protein-protein interactions.

Polarized left-handed extraordinary optical transmission of subterahertz waves

In this paper we design and measure a metamaterial polarizing device working in the sub-terahertz range. The polarizer is based on a modified version of our previous miniaturized Stacked Hole Array (SHA) structure, an arrangement that combines Extraordinary Optical Transmission (EOT) and Left-Handed Metamaterial (LHM) propagation even under Fresnel illumination. Here, we use a self complementary screen by connecting the holes of an EOT structure. Importantly, EOT remains and simultaneously total reflection is obtained for the orthogonal component. Moreover, by computing the dispersion diagram, we demonstrate that LHM propagation can be achieved for the principal polarization within the stop band of the orthogonal component, which propagates in other bands as a standard forward wave. Finally, we check our conjectures by measuring the transmission and reflection coefficients of screens milled on a low-loss microwave substrate. Measurements have been taken for 1 to 6 stacked wafers and they show clearly that the stack acts as a polarizer with left-handed characteristic. Our results open the way to design of novel polarization control metamaterials at Terahertz wavelengths.

Genetic Modifier Screens in Drosophila Demonstrate a Role for Rho1 Signaling in Ecdysone-Triggered Imaginal Disc Morphogenesis

Drosophila adult leg development provides an ideal model system for characterizing the molecular mechanisms of hormone-triggered morphogenesis. A pulse of the steroid hormone ecdysone at the onset of metamorphosis triggers the rapid transformation of a flat leg imaginal disc into an immature adult leg, largely through coordinated changes in cell shape. In an effort to identify links between the ecdysone signal and the cytoskeletal changes required for leg morphogenesis, we performed two large-scale genetic screens for dominant enhancers of the malformed leg phenotype associated with a mutation in the ecdysone-inducible broad early gene (br1). From a screen of >750 independent deficiency and candidate mutation stocks, we identified 17 loci on the autosomes that interact strongly with br1. In a complementary screen of approximately 112,000 F1 progeny of EMS-treated br1 animals, we recovered 26 mutations that enhance the br1 leg phenotype [E(br) mutations]. Rho1, stubbloid, blistered (DSRF), and cytoplasmic Tropomyosin were identified from these screens as br1-interacting genes. Our findings suggest that ecdysone exerts its effects on leg morphogenesis through a Rho1 signaling cascade, a proposal that is supported by genetic interaction studies between the E(br) mutations and mutations in the Rho1 signaling pathway. In addition, several E(br) mutations produce unexpected defects in midembryonic morphogenetic movements. Coupled with recent evidence implicating ecdysone signaling in these embryonic morphogenetic events, our results suggest that a common ecdysone-dependent, Rho1-mediated regulatory pathway controls morphogenesis during the two major transitions in the life cycle, embryogenesis and metamorphosis.

Strings in homogeneous gravitational waves and null holography

Homogeneous gravitational wave backgrounds arise as infinite momentum limits of many geometries with a well-understood holographic description. General global aspects of these geometries are discussed. Using exact CFT techniques, strings in pp-wave backgrounds supported by a Neveu-Schwarz flux are quantized. As in Euclidean $AdS_3$, spectral flow and associated long strings are shown to be crucial in obtaining a complete spectrum. Holography is investigated using conformally flat coordinates analogous to those of the Poincar\'e patch in AdS. It is argued that the holographic direction is the light-cone coordinate $u$, and that the holographic degrees of freedom live on a codimension-one screen at fixed $u$. The usual conformal symmetry on the boundary is replaced by a representation of a Heisenberg-type algebra $H_D\times H_D$, hinting at a new class of field theories realizing this symmetry. A sample holographic computation of 2 and 3-point functions is provided and Ward identities are derived. A complementary screen at fixed $v$ is argued to be necessary in order to encode the vacuum structure.

A functional screen identifies hDRIL1 as an oncogene that rescues RAS-induced senescence.

Primary fibroblasts respond to activated H-RAS(V12) by undergoing premature arrest, which resembles replicative senescence. This irreversible 'fail-safe mechanism' requires p19(ARF), p53 and the Retinoblastoma (Rb) family: upon their disruption, RAS(V12)-expressing cells fail to undergo senescence and continue to proliferate. Similarly, co-expression of oncogenes such as c-MYC or E1A rescues RAS(V12)-induced senescence. To identify novel genes that allow escape from RAS(V12)-induced senescence, we designed an unbiased, retroviral complementary DNA library screen. We report on the identification of DRIL1, the human orthologue of the mouse Bright and Drosophila dead ringer transcriptional regulators. DRIL1 renders primary murine fibroblasts unresponsive to RAS(V12)-induced anti-proliferative signalling by p19(ARF)/p53/p21(CIP1), as well as by p16(INK4a). In this way, DRIL1 not only rescues RAS(V12)-induced senescence but also causes these fibroblasts to become highly oncogenic. Furthermore, DRIL1 immortalizes mouse fibroblasts, in the presence of high levels of p16(INK4a). Immortalization by DRIL1, whose product binds the pRB-controlled transcription factor E2F1 (ref. 8), is correlated with induction of E2F1 activity. Correspondingly, DRIL1 induces the E2F1 target Cyclin E1, overexpression of which is sufficient to trigger escape from senescence. Thus, DRIL1 disrupts cellular protection against RAS(V12)-induced proliferation downstream of the p19(ARF)/p53 pathway.

Numerical solution for diffraction of an electromagnetic wave by slits in a perfectly conducting screen

A method is described for computing, from first principles, the diffraction pattern from an array of parallel slits in a thin perfectly conducting screen. The incident wave is plane and has an arbitrary direction. Babinet9s principle relates this problem to that of the complementary screen, an array of parallel strips. It is already known that the whole problem may be reduced to the solution of two integral equations. Two suitable ones are derived and solved numerically for the case of one or two strips or slits; the extension to more than two is then obvious. The interaction between the strips is clearly seen in the distributions of surface currents induced in them; these are shown graphically for different polarizations, widths and angles of incidence. The field distributions very near to the screen are presented as contour maps and compared with microwave measurements made by the modulated scatterer method; the far field is also examined. For wide strips or slits an alternative approximate numerical method is suggested.

Babinet’s principle for elastic waves: A numerical test

Babinet’s principle states that the diffracted fields from complementary screens are the negative of each other. In electromagnetics, Babinet’s principle for infinitely thin perfectly conducting complementary screens implies that the sum, beyond the screen plane, of the electric and the magnetic fields (adjusting physical dimensions) equals the incident (unscreened) electric field. A test of the principle for the elastodynamic case was made using numerical calculations, and the results demonstrate that Babinet’s principle holds quite well for complementary plane screens with contrasting boundary conditions; that is, the complementary screen of a stress-free screen is a rigid screen with openings where the original stress-free screen existed, and vice versa. The results are exact in an anisotropic SH case; for the P–SV case, the diffracted waves, PdP, SdS, PdS, and SdP satisfy the principle exactly, while the refracted waves, PdPrSc and SdPrSc, do not satisfy the principle at all (these waves are generally much smaller than the PdS and SdP waves). Diffracted surface waves also do not satisfy the principle. The numerical method is based on a domain-decomposition technique that assigns a different mesh to each side of the screen plane. The effects of the screens on wave propagation are modeled through the boundary conditions, requiring a special boundary treatment based on characteristic variables. The algorithm solves the velocity/stress wave equations and is based on a Fourier/Chebyshev differential operator.

Isolation and characterization of new alleles of the cyclin-dependent kinase gene CDC28 with cyclin-specific functional and biochemical defects.

The G1 cyclin Cln2 negatively regulates the mating-factor pathway. In a genetic screen to identify factors required for this regulation, we identified an allele of CDC28 (cdc28-csr1) that blocked this function of Cln2. Cln2 immunoprecipitated from cdc28-csr1 cells was completely defective in histone H1 kinase activity, due to defects in Cdc28 binding and activation by Cln2. In contrast, Clb2-associated H1 kinase and Cdc28 binding was normal in immunoprecipitates from these cells. cdc28-csr1 was significantly deficient in other aspects of genetic interaction with Cln2. The cdc28-csr1 mutation was determined to be Q188P, in the T loop distal to most of the probable Cdk-cyclin interaction regions. We performed random mutagenesis of CDC28 to identify additional alleles incapable of causing CLN2-dependent mating-factor resistance but capable of complementing cdc28 temperature-sensitive and null alleles. Two such mutants had highly defective Cln2-associated kinase, but, surprisingly, two other mutants had levels of Cln2-associated kinase near to wild-type levels. We performed a complementary screen for CDC28 mutants that could cause efficient Cln2-dependent mating-factor resistance but not complement a cdc28 null allele. Most such mutants were found to alter residues essential for kinase activity; the proteins had little or no associated kinase activity in bulk or in association with Cln2. Several of these mutants also functioned in another assay for CLN2-dependent function not involving the mating-factor pathway, complementing the temperature sensitivity of a cln1 cln3 cdc28-csr1 strain. These results could indicate that Cln2-Cdc28 kinase activity is not directly relevant to some CLN2-mediated functions. Mutants of this sort should be useful in differentiating the function of Cdc28 complexed with different cyclin regulatory subunits.

A strategy for screening anti-tumor drugs utilizing oncogenes encoded in retroviral vectors

A novel strategy for isolating potential anti-tumor drugs is presented. It is predicated on the idea that future anti-tumor drugs will be specific inhibitors of the signal-transduction pathways responsible for cell proliferation. Briefly, retroviral vectors are used to introduce focus-forming oncogenes into a test population of target cells, which are grown to confluence and treated with signal-transduction inhibitors. The inhibitors are screened for the ability to suppress the development of transformed foci without killing the confluent monolayer of non-transformed quiescent cells. For this work, a panel of inhibitors was first screened against the oncogene ras. The protein kinase C (PKC) inhibitor CGP 41251 and the protein tyrosine kinase (PTK) inhibitor CGP 45047 suppressed ras-induced focus formation and left a viable monolayer of quiescent cells. Focus inhibition was reversible; conversely, drug addition to developing foci retarded further expansion. CGP 41251 generally blocked proliferation of ras or control cells, suggesting that oncogenes cannot substitute for PKC. PTK inhibitors erbstatin and CGP 520 and phosphatase inhibitor okadaic acid failed to inhibit focus formation at concentrations toxic to the monolayer. Lavendustin A and CGP 47778A showed neither focus inhibition nor toxicity. In the complementary screen, a single inhibitor (CGP 41251) was tested against several oncogenes, including src, raf and polyomavirus middle T antigen. Focus formation by all oncogenes was suppressed. The strategy has several advantages over current drug-screening assays, and it can be adapted to large-scale screening with many drugs and many oncogenes.

A strategy for screening anti‐tumor drugs utilizing oncogenes encoded in retroviral vectors

A novel strategy for isolating potential anti-tumor drugs is presented. It is predicated on the idea that future anti-tumor drugs will be specific inhibitors of the signal-transduction pathways responsible for cell proliferation. Briefly, retroviral vectors are used to introduce focus-forming oncogenes into a test population of target cells, which are grown to confluence and treated with signal-transduction inhibitors. The inhibitors are screened for the ability to suppress the development of transformed foci without killing the confluent monolayer of non-transformed quiescent cells. For this work, a panel of inhibitors was first screened against the oncogene ras. The protein kinase C (PKC) inhibitor CGP 41251 and the protein tyrosine kinase (PTK) inhibitor CGP 45047 suppressed ras-induced focus formation and left a viable monolayer of quiescent cells. Focus inhibition was reversible; conversely, drug addition to developing foci retarded further expansion. CGP 41251 generally blocked proliferation of ras or control cells, suggesting that oncogenes cannot substitute for PKC. PTK inhibitors erbstatin and CGP 520 and phosphatase inhibitor okadaic acid failed to inhibit focus formation at concentrations toxic to the monolayer. Lavendustin A and CGP 47778A showed neither focus inhibition nor toxicity. In the complementary screen, a single inhibitor (CGP 41251) was tested against several oncogenes, including src, raf and polyomavirus middle T antigen. Focus formation by all oncogenes was suppressed. The strategy has several advantages over current drug-screening assays, and it can be adapted to large-scale screening with many drugs and many oncogenes. © 1996 Wiley-Liss, Inc.

A neural tetraspanin, encoded by late bloomer, that facilitates synapse formation.

Upon contacting its postsynaptic target, a neuronal growth cone transforms into a presynaptic terminal. A membrane component on the growth cone that facilitates synapse formation was identified by means of a complementary DNA-based screen followed by genetic analysis. The late bloomer (lbl) gene in Drosophila encodes a member of the tetraspanin family of cell surface proteins. LBL protein is transiently expressed on motor axons, growth cones, and terminal arbors. In lbl mutant embryos, the growth cone of the RP3 motoneuron contacts its target muscles, but synapse formation is delayed and neighboring motoneurons display an increase in ectopic sprouting.

Simple formulas for transmission through periodic metal grids or plates

A simple closed-form approximate solution is given for the transmission coefficient of a normally incident electromagnetic plane wave through a screen made of periodic metal grids (inductive screen), or made of metal plates (the complementary capacitive screen). Explicit formulas are also presented for cascading screens and dielectric slabs. When compared with the exact solution, our approximate simple formulas show good numerical accuracy.

THE PERTURBATION THEORY FOR THE FIRST-ORDER APPROXIMATION OF THE DIFFRACTION PROBLEMS

Applying the perturbation theory to Helmholtz's equations of spherical and sphero-conal systems of coordinates, the author deduces the expressions for the perturbation terms of diffraction due to a plane angular sector with its angle nearly equal to π and those due to an elliptic cone with θ nearly equal to π/2 respectively. Then, following the principle in [10], substituting the diffracting plane disk of arbitrary form by its inscribed polygonal disk and the diffracting surface of arbitrary form by a surface which consists of many small inscribed elliptic cones of the original surface, we make the summations of the perturbation terms for the sectors of the polygon and those of the tops of the small cones respectively. In the limiting case these two summations become integrals, hence the diffraction fields of first-order due to the disk (thereof, the complementary plane screen with a hole) and the surface of arbitrary forms are derived respectively.

A theory of the diffraction of an electromagnetic wave by a plane screen

The problem of the diffraction of an arbitrarily polarized electromagnetic plane wave by a perfectly conducting plane screen of infinitesimal thickness is reduced to scalar problems by the method known from earlier papers. The wave field diffracted either by a screen of infinite extent which is perforated by an aperture of arbitrary size and shape or by a complementary screen is then described by means of the bra-vectors, ket-vectors and linear operators introduced by the present author in one of his preceding papers.