Screening Validation Research Articles

GATA1-Centered Genetic Network on Chromosome 21 Drives Down Syndrome Acute Megakaryoblastic Leukemia

Children with trisomy 21 (Down syndrome, DS) are predisposed to develop acute megakaryoblastic leukemia (DS-AMKL) as well as the antecedent transient leukemia (DS-TL). Mutations in the transcription factor GATA1 -leading to the exclusive expression of the shorter GATA1 isoform (GATA1s)- are present in nearly all children with DS-AMKL and DS-TL. GATA1s is both essential and sufficient to cause DS-TL in synergy with trisomy 21.To elucidate how the presence of an extra copy of chromosome 21 (hsa21) perturbs fetal hematopoiesis to provide a GATA1s-sensitive background during trisomy 21-associated leukemogenesis, we integrated an RNAi viability screening (512 shRNAmirs against 210 genes on hsa21) and a proteomics approach creating an hsa21 oncogenic network centered on GATA1s. shRNA-mediated knock-down of 42 genes conferred a profound selective growth disadvantage in DS-AMKL cell lines (CMK and CMY). A secondary functional validation screening confirmed 8 genes to specifically affect proliferation, cell viability, apoptosis or differentiation in GATA1s/trisomy-associated leukemia; whereas expression of 9 genes was also essential for proliferation and survival of erythroleukemia (K562) and non-DS-AMKL (M07) cell lines.Gain- and loss-of-function studies of 12 selected candidates (8 GATA1s/trisomy-specific oncogenes plus 4 global oncogenes) in CD34+ hematopoietic stem and progenitor cells (HSPCs) uncovered their regulatory function during megakaryopoiesis, erythropoiesis and myelopoiesis. Knockdown of four genes (USP25, BACH1, U2AF1 and C21orf33) inhibited megakaryocytic and erythroid in vitro differentiation, while enhancing myeloid differentiation. Inversely, ectopic expression of six genes (C21orf33, CHAF1B, IFNGR2, WDR4, RUNX1 or GABPA) resulted in a switch from erythroid to megakaryocytic differentiation. These 12 candidate genes acted synergistically to enhance the self-renewal efficiency of murine fetal liver cells in vitro. Pooled transduction of these genes increased the replating efficiency (more than 5 rounds) of fetal liver HSPCs whereas the colony-forming capacity was lost after second replating in the empty vector control. Further, 9 out of 12 candidate genes were overexpressed in DS-AMKL patient samples (n=23) compared to non-DS-AMKL (n=37; 1.3-fold to 2-fold) underscoring their relevance for the pathogenesis of DS-AMKL.Using an in vivo biotinylation approach to study the protein-protein interaction in DS-AMKL cells, we showed that bioGATA1 is associated with protein-complexes of 10 different hsa21-oncogenes, which are involved in splicing, deubiquitination and transcriptional regulation. Direct interactions with several of these factors are perturbed in N-terminal truncated GATA1s.Thus, we deciphered a complex interactive network on hsa21 around GATA1 positively regulating megakaryopoiesis. Deregulation of this network results in synergistic effects on hematopoietic differentiation, which can promote transformation of GATA1s-mutated fetal hematopoietic progenitor cells. DisclosuresNo relevant conflicts of interest to declare.

Characterization of oncogenes on chromosome 21 identified by shRNA-based viability screening

Children with trisomy 21 (Down syndrome, DS) are predisposed to develop acute megakaryoblastic leukemia (DS-AMKL) as well as the antecedent transient leukemia (DS-TL). Mutations in the transcription factor GATA1 have been found in nearly all children with DS-AMKL and DS-TL, but not in other malignancies. Recent whole genome sequencing efforts suggested that the interplay of trisomy 21 and GATA1s-mutation is sufficient to cause DS-TL. To decipher the deregulated oncogenic gene network on hsa21, we conducted a lentiviral shRNA-based viability screening using GATA1s-mutated DS-AMKL cell line CMK as well as non-DS-AML cell lines (K562, M-07). Knock-down of 42 genes conferred a profound selective growth disadvantage in DS-AMKL cell lines. 31 candidate genes are located in chromosomal region 21q22.1-21q22.3. 11 (out of 14 tested) were overexpressed in DS-AMKL compared to non-DS-AMKL. A secondary functional validation screening revealed that the potential oncogenes participate in different cellular processes affecting proliferation, cell viability, apoptosis or differentiation. To further delineate the impact of 11 selected candidates on normal hematopoiesis, we characterized their effects in gain- and loss-of-function studies using CD34+ hematopoietic stem and progenitor cells (HSPCs). Knockdown of four genes (USP25, BACH1, U2AF1 and C21orf33) inhibited megakaryocytic and erythroid in vitro differentiation, while enhancing myeloid differentiation. The myeloid differentiation bias was also observed for ATP5O and C21orf45. The opposite effect was observed in gain-of-function studies. Ectopic expression of six genes (U2AF1, C21orf33, IFNGR2, WDR4 or GABPA) resulted in a switch from erythroid to megakaryocytic differentiation. Thus, we propose a complex interactive network located on hsa21. Deregulation of this network might result in synergistic effects on hematopoietic differentiation, which promotes transformation of GATA1s-mutated fetal hematopoietic progenitor cells.

Validation and target gene screening of hsa-miR-205 in lung squamous cell carcinoma

Background Lung cancers are classified as squamous cell carcinoma (SQ), adenocarcinoma (AC) and small cell lung carcinoma (SCLC). SQ is the major subtype of lung cancer. Currently, there are no targeted therapies for SQ due to lack of understanding its driving oncogenes. In this study, we validated an SQ specific biomarker hsa-miR-205 in Chinese patients with lung cancer and screened its candidate target genes for further functional studies to enrich knowledge in SQ target therapies. Methods Quantitative reverse-transcription PCR (quantitative RT-PCR) was performed on 197 macro-dissected (cancerous cells >75%) surgical lung tissues (45 SQ, 44 AC, 54 SCLC and 54 adjacent normal tissues) to validate the expression profiles of miR-205. Furthermore, the targets of this microRNA were predicted through the gateway miRecords and mapped to lung cancer-associated pathways using the KEGG (Kyoto Encyclopedia of Genes and Genomes) database. Then quantitative RT-PCR was performed on an independent cohort of 44 snap-frozen surgical lung tissues to concurrently assess the expression profiles of miR-205 and its 52 putative targeted genes. Results MicroRNA-205 yielded high diagnostic accuracy in discriminating SQ from AC with an area under the curve (AUC) of 0.985, and discriminating SQ from SCLC with an AUC of 0.978 in formalin-fixed paraffin-embedded (FFPE) surgical lung tissues. Predicted targets of miR-205 were associated with 52 key members of lung cancer signaling pathways. Ten target genes (ACSL1, AXIN2, CACNA2D2, FOXO3, PPP1R3A, PRKAG3, RUNX1, SMAD4, STK3 and TBL1XR1) were significantly down-regulated in SQ and had a strong negative correlation with miR-205, while one target gene (CDH3) was up-regulated in SQ and exhibited a strong positive correlation with miR-205. Conclusions We confirmed the high diagnostic accuracy of miR-205 in discriminating SQ from AC and SCLC in Chinese patients. Moreover, we identified 11 significant target genes of miR-205 which could be used for further functional studies as the basis for the development of SQ targeted therapies.

Development, validation and pilot screening of an in vitro multi-cellular three-dimensional cancer spheroid assay for anti-cancer drug testing

It has been demonstrated that two-dimensional (2D) monolayer cancer cell proliferation assay for anti-cancer drug screening is a very artificial model and cannot represent the characteristics of three-dimensional (3D) solid tumors. The multi-cellular in vitro 3D tumor spheroid model is of intermediate complexity, and can provide a bridge to the gap between the complex in vivo tumors and simple in vitro monolayer cell cultures. In this study, a simple and cost-effective cancer 3D spheroid assay suitable for small molecule anti-cancer compound screening was developed, standardized and validated on H292 non-small lung cancer cell line. A pilot screening with this assay was performed utilizing a compound library consisting of 41 anti-cancer agents. The traditional 2D monolayer cell proliferation assay was also performed with the same cell line and compounds. A correlational study based on the IC50 values from the 2D and 3D assays was conducted. There is low correlation with the two sets of biological data, suggesting the two screening methods provide different information regarding the potency of the tested drug candidates.

Characterization of oncogenes on chromosome 21 identified by shRNA-based viability screening

Children with trisomy 21 (Down syndrome, DS) are predisposed to develop acute megakaryoblastic leukemia (DS-AMKL) as well as the antecedent transient leukemia (DS-TL). Mutations in the transcription factor GATA1 have been found in nearly all children with DS-AMKL and DS-TL, but not in other malignancies. Recent whole genome sequencing efforts suggested that the interplay of trisomy 21 and GATA1s-mutation is sufficient to cause DS-TL. To decipher the deregulated oncogenic gene network on hsa21, we conducted a lentiviral shRNA-based viability screening using GATA1s-mutated DS-AMKL cell line CMK as well as non-DS-AML cell lines (K562, M-07). Knock-down of 42 genes conferred a profound selective growth disadvantage in DS-AMKL cell lines. 31 candidate genes are located in chromosomal region 21q22.1-21q22.3. 11 (out of 14 tested) were overexpressed in DS-AMKL compared to non-DS-AMKL. A secondary functional validation screening revealed that the potential oncogenes participate in different cellular processes affecting proliferation, cell viability, apoptosis or differentiation. To further delineate the impact of 11 selected candidates on normal hematopoiesis, we characterized their effects in gain- and loss-of-function studies using CD34+ hematopoietic stem and progenitor cells (HSPCs). Knockdown of four genes (USP25, BACH1, U2AF1 and C21orf33) inhibited megakaryocytic and erythroid in vitro differentiation, while enhancing myeloid differentiation. The myeloid differentiation bias was also observed for ATP5O and C21orf45. The opposite effect was observed in gain-of-function studies. Ectopic expression of six genes (U2AF1, C21orf33, IFNGR2, WDR4 or GABPA) resulted in a switch from erythroid to megakaryocytic differentiation. Thus, we propose a complex interactive network located on hsa21. Deregulation of this network might result in synergistic effects on hematopoietic differentiation, which promotes transformation of GATA1s-mutated fetal hematopoietic progenitor cells.

Melamine and Cyanuric Acid in Foodstuffs and Pet Food: Method Validation and Sample Screening

Hazardous levels of melamine in food and feed products have been of great concern after the outbreak of contamination reported in Chinese commodities in 2008. Despite the existence of several analytical methods for melamine (MEL) detection in food, few provide a full validation data set, especially when MEL and cyanuric acid (CYA) are analyzed simultaneously. The aim of this study was to validate an analytical methodology for MEL and CYA analysis in foodstuffs by GC-MS after trimethylsilylation with N,O-bis(trimethylsilyl)trifluoroacetamide. Linearity was obtained in the range of 0.4 to 800 mg/kg for both compounds, with limits of detection (LODs) of 0.15 and 0.05 mg/kg for MEL and CYA, respectively. Screening in 20 food products [3 soya milk powder, 1 baby milk powder, 3 soya powder, 13 diversified cookies and biscuits (8 from China and 2 from Portugal), and 3 dog food) revealed MEL incidence in 55% of the cases, with a maximum concentration of 3.4 mg/kg. CYA was not detected in all samples.

Chemiluminescent Detection of Carbohydrates in the Tumoral Breast Diseases

Nowadays, there is an increase of investigations into the fibroadenoma, mainly because some studies have shown that the occurrence of fibroadenoma is linked to an increased risk of developing breast carcinoma. Currently, the chemiluminescence biomarkers are applied for validation methods and screening. Here, a lectin chemiluminescence is proposed as new histochemistry method to identify carbohydrates in mammary tumoral tissues. The lectins concanavalin A (Con A) and peanut agglutinin (PNA) conjugated to acridinium ester were used to characterize the glycocode of breast tissues: normal, fibroadenoma, and invasive duct carcinoma (IDC). The lectin chemiluminescence expressed in relative light units (RLU) was higher in fibroadenoma and IDC than in normal tissue for both lectins tested. The relationship RLU emission versus tissue area described a linear and hyperbolic curve for IDC and fibroadenoma, respectively, using Con A whereas hyperbolic curves for both transformed tissues using PNA. RLU was abolished by inhibiting the interaction between tissues and lectins using their specific carbohydrates: methyl-α-D: -mannoside (Con A) and galactose (PNA). The intrinsic fluorescence emission did not change with combination of the lectins (Con A/PNA) to the acridinium ester for hydrophobic residues. These results represent the lectin chemiluminescence as an alternative of histochemistry method for tumoral diagnosis in the breast.

Analysis of banana fruit-specific promoters using transient expression in embryogenic cells of banana cultivar Robusta (AAA Group)

The study reports the transient expression of gusA gene in embryogenic cells using three banana derived fruit-specific promoters. Banana embryogenic cells were transformed with a pCAMBIA-1301 derived plasmid construct harboring gusA gene driven by either chitinase, glucanase or expansin promoters derived from banana. The transient expression of β-glucuronidase was studied 5 days after co-cultivation with Agrobacterium harboring the expression plasmids. The transformed embryogenic cells were treated with different inducers of ethylene such as ethephon, methyl jasmonate, methyl salicylate, abscisic acid and indole acetic acid. The maximum expression of 64099.78 pmoles 4-MU/h/mg total protein was noted with expansin promoter when the cells were treated with the combination of ethephon (0.25 mM) and MJ (10 mM). The results suggest that these promoters can be used to achieve fruit-specific expression of useful transgenes in banana. The results should prove to be an important guide for short term expression studies for promoter validation and gene screening.

Open Innovation for Phenotypic Drug Discovery: The PD2 Assay Panel

Phenotypic lead generation strategies seek to identify compounds that modulate complex, physiologically relevant systems, an approach that is complementary to traditional, target-directed strategies. Unlike gene-specific assays, phenotypic assays interrogate multiple molecular targets and signaling pathways in a target "agnostic" fashion, which may reveal novel functions for well-studied proteins and discover new pathways of therapeutic value. Significantly, existing compound libraries may not have sufficient chemical diversity to fully leverage a phenotypic strategy. To address this issue, Eli Lilly and Company launched the Phenotypic Drug Discovery Initiative (PD(2)), a model of open innovation whereby external research groups can submit compounds for testing in a panel of Lilly phenotypic assays. This communication describes the statistical validation, operations, and initial screening results from the first PD(2) assay panel. Analysis of PD(2) submissions indicates that chemical diversity from open source collaborations complements internal sources. Screening results for the first 4691 compounds submitted to PD(2) have confirmed hit rates from 1.6% to 10%, with the majority of active compounds exhibiting acceptable potency and selectivity. Phenotypic lead generation strategies, in conjunction with novel chemical diversity obtained via open-source initiatives such as PD(2), may provide a means to identify compounds that modulate biology by novel mechanisms and expand the innovation potential of drug discovery.

High-throughput Screening Uncovers a Compound That Activates Latent HIV-1 and Acts Cooperatively with a Histone Deacetylase (HDAC) Inhibitor

Current antiretroviral therapy (ART) provides potent suppression of HIV-1 replication. However, ART does not target latent viral reservoirs, so persistent infection remains a challenge. Small molecules with pharmacological properties that allow them to reach and activate viral reservoirs could potentially be utilized to eliminate the latent arm of the infection when used in combination with ART. Here we describe a cell-based system modeling HIV-1 latency that was utilized in a high-throughput screen to identify small molecule antagonists of HIV-1 latency. A more detailed analysis is provided for one of the hit compounds, antiviral 6 (AV6), which required nuclear factor of activated T cells for early mRNA expression while exhibiting RNA-stabilizing activity. It was found that AV6 reproducibly activated latent provirus from different lymphocyte-based clonal cell lines as well as from latently infected primary resting CD4(+) T cells without causing general T cell proliferation or activation. Moreover, AV6 complemented the latency antagonist activity of a previously described histone deacetylase (HDAC) inhibitor. This is a proof of concept showing that a high-throughput screen employing a cell-based model of HIV-1 latency can be utilized to identify new classes of compounds that can be used in concert with other persistent antagonists with the aim of viral clearance.

Abstract 4277: High throughput 3D tumor spheroid assays for target validation and drug evaluation

Abstract Background: Standard 2D monolayer cultures used for target validation and drug screening studies do not adequately address the complexity of in vivo tumor pathophysiology. Although 3D tumor spheroids better represent a tumor microenvironment (with gradients of proliferation, oxygenation and drug access) they have not been routinely used in high-throughput (HTS) mode. The aim of the present study was to develop a suite of in vitro 3D spheroid-based assays to rapidly and accurately quantify key aspects of the malignant phenotype: growth, motility, invasion and angiogenesis. Methods: We established a novel, standardised method for the generation of tumor spheroids in suspension using ultra-low attachment (ULA) 96-well plates. Spheroids were characterized in terms of growth kinetics, haptotaxis on extracellular matrix (ECM) and endothelial cell (EC) monolayers, invasion into MatrigelTM and co-culture with embryoid bodies (EB) to model tissue invasion/angiogenesis. Quantitative data were obtained by image analysis using a Celigo cytometer or microscopy. Cell viability was determined by the CellTiter Glo assay for direct comparison of 2D and 3D cultures. Highly malignant human adult (U87MG) and pediatric (KNS42) gliomas and breast carcinoma (MDA MB 231) spheroids were treated with inhibitors of HSP90 (17-AAG) and PI3K (PI-103) to demonstrate the power of the techniques. Results: Our standardized protocol generates single spheroids/well which are reproducible in size and easy to handle. Images are obtained at intervals over 14 days for growth kinetics or 48-96 hours for functional assays. The Celigo cytometer enables rapid, multiparametric real-time analyses, and samples can be collected to measure target protein expression, drug levels and pharmacodynamic effects. Dose-dependent inhibition of 3D spheroids was readily demonstrated using the targeted agents 17-AAG and PI-103. All models also showed extensive migration and invasion mirroring their in-vivo behaviour; interestingly U87MG and KNS42 showed different patterns of dissemination: widely dispersed or contiguous respectively. MDA MB 231 migration and invasion were inhibited with sub GI50 concentrations of 17-AAG. GFP-labeled U87MG and MDA MB 231 tumor spheroids co-cultured with EBs rapidly attached and coalesced, providing a model to study simultaneous tissue invasion and angiogenesis. Direct comparisons of cell viability in 2D and 3D showed that U87MG and MDA MB 231 are more resistant to 17-AAG in 3D, conversely U87MG was more sensitive to PI-103 in 3D. Conclusions: We have developed a unique repertoire of novel 3D multiplate assays reflecting key aspects of malignant cell behaviour and able to deliver rapid and reproducible results. We provide evidence that our methods potentially enhance target selection and the triaging of drug candidates prior to in vivo studies. 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 4277. doi:10.1158/1538-7445.AM2011-4277

Live Cell in Vitro and in Vivo Imaging Applications: Accelerating Drug Discovery

Dynamic regulation of specific molecular processes and cellular phenotypes in live cell systems reveal unique insights into cell fate and drug pharmacology that are not gained from traditional fixed endpoint assays. Recent advances in microscopic imaging platform technology combined with the development of novel optical biosensors and sophisticated image analysis solutions have increased the scope of live cell imaging applications in drug discovery. We highlight recent literature examples where live cell imaging has uncovered novel insight into biological mechanism or drug mode-of-action. We survey distinct types of optical biosensors and associated analytical methods for monitoring molecular dynamics, in vitro and in vivo. We describe the recent expansion of live cell imaging into automated target validation and drug screening activities through the development of dedicated brightfield and fluorescence kinetic imaging platforms. We provide specific examples of how temporal profiling of phenotypic response signatures using such kinetic imaging platforms can increase the value of in vitro high-content screening. Finally, we offer a prospective view of how further application and development of live cell imaging technology and reagents can accelerate preclinical lead optimization cycles and enhance the in vitro to in vivo translation of drug candidates.

Detecting interactions with membrane proteins using a membrane two-hybrid assay in yeast

The biological function of proteins may be predicted by identification of their interacting partners, and one of the major goals of the postgenomic era is the mapping of protein interaction networks. Membrane proteins are of particular interest because of their role in disease and because of their prevalence as major pharmaceutical targets. Unfortunately, because of their hydrophobic nature, they have long been difficult to study in a high-throughput format. A powerful technology recently developed to facilitate the characterization of membrane protein interactions is the membrane yeast two-hybrid (MYTH) assay. MYTH adapts the principle of split ubiquitin for use as a potent in vivo sensor of protein-protein interactions, allowing large-scale screening for interactors of full-length membrane proteins, from a range of organisms, using Saccharomyces cerevisiae as a host. In this article, we describe a protocol for MYTH bait generation, validation and library screening. The entire MYTH procedure can generally be completed in 4-6 weeks.

Identification of Tumor-associated Autoantigens for the Diagnosis of Colorectal Cancer in Serum Using High Density Protein Microarrays

There is a mounting evidence of the existence of autoantibodies associated to cancer progression. Antibodies are the target of choice for serum screening because of their stability and suitability for sensitive immunoassays. By using commercial protein microarrays containing 8000 human proteins, we examined 20 sera from colorectal cancer (CRC) patients and healthy subjects to identify autoantibody patterns and associated antigens. Forty-three proteins were differentially recognized by tumoral and reference sera (p value <0.04) in the protein microarrays. Five immunoreactive antigens, PIM1, MAPKAPK3, STK4, SRC, and FGFR4, showed the highest prevalence in cancer samples, whereas ACVR2B was more abundant in normal sera. Three of them, PIM1, MAPKAPK3, and ACVR2B, were used for further validation. A significant increase in the expression level of these antigens on CRC cell lines and colonic mucosa was confirmed by immunoblotting and immunohistochemistry on tissue microarrays. A diagnostic ELISA based on the combination of MAPKAPK3 and ACVR2B proteins yielded specificity and sensitivity values of 73.9 and 83.3% (area under the curve, 0.85), respectively, for CRC discrimination after using an independent sample set containing 94 sera representative of different stages of progression and control subjects. In summary, these studies confirmed the presence of specific autoantibodies for CRC and revealed new individual markers of disease (PIM1, MAPKAPK3, and ACVR2B) with the potential to diagnose CRC with higher specificity and sensitivity than previously reported serum biomarkers.

Systematic analysis of a simple adaptor protein PDZK1: ligand identification, interaction and functional prediction of complex

PDZK1 is a simple adaptor protein with four protein interaction PDZ domains, but without any other known functional domains. Here, we used yeast two‐hybrid screening of a random peptide library and high‐throughput validation screening of a specialized PDZ ligand candidate library to systematically and comprehensively identify PDZK1 ligands. The potential functional associations of the ligands were predicted by functional annotations from a MILANO literature search and subcellular localizations. The ligands were considered more likely to be functionally associated if they had similar patterns of functions or closely related functions. For some functionally associated ligand pairs, interaction with one ligand was found to be influenced by another ligand in a yeast three‐hybrid system. Many G‐protein signaling pathway‐related proteins were found to interact with PDZK1, and they were likely to be functionally associated with transporters based on their closely related functions. This strategy can be extended to the study of other adaptor proteins that contain peptide‐binding domains.

Systematic Analysis of a Simple Adaptor Protein PDZK1: Ligand Identification, Interaction and Functional Prediction of Complex

PDZK1 is a simple adaptor protein with four protein interaction PDZ domains, but without any other known functional domains. Here, we used yeast two-hybrid screening of a random peptide library and high-throughput validation screening of a specialized PDZ ligand candidate library to systematically and comprehensively identify PDZK1 ligands. The potential functional associations of the ligands were predicted by functional annotations from a MILANO literature search and subcellular localizations. The ligands were considered more likely to be functionally associated if they had similar patterns of functions or closely related functions. For some functionally associated ligand pairs, interaction with one ligand was found to be influenced by another ligand in a yeast three-hybrid system. Many G-protein signaling pathway-related proteins were found to interact with PDZK1, and they were likely to be functionally associated with transporters based on their closely related functions. This strategy can be extended to the study of other adaptor proteins that contain peptide-binding domains.

Image-based evaluation of the molecular events underlying HC11 mammary epithelial cell differentiation

We have developed an image-based technique for signal pathway analysis, target validation, and compound screening related to mammary epithelial cell differentiation. This technique used the advantages of optical imaging and the HC11-Lux model system. The HC11-Lux cell line is a subclone of HC11 mammary epithelial cells transfected stably with a luciferase construct of the β-casein gene promoter (p-344/-1βc-Lux). The promoter activity was imaged optically in real time following lactogenic induction. The imaging signal intensity was closely correlated with that measured using a luminometer following protein extraction ( R = 0.99, P < 0.0001) and consistent with the messenger RNA (mRNA) level of the endogenous β -casein gene. Using this technique, we examined the roles of JAK2/Stat5A, Raf-1/MEK/MAKP, and PI3K/Akt signal pathways with respect to differentiation. The imaging studies showed that treatment of the cells with epidermal growth factor (EGF), AG490 (JAK2-specific inhibitor), and LY294002 (PI3K-specific inhibitor) blocked lactogenic differentiation in a dose-dependent manner. PD98059 (MEK-specific inhibitor) could reverse EGF-mediated differentiation arrest. These results indicate that these pathways are essential in cell differentiation. This simple, sensitive, and reproducible technique permits visualization and real-time evaluation of the molecular events related to milk protein production. It can be adopted for high-throughput screening of small molecules for their effects on mammary epithelial cell growth, differentiation, and carcinogenesis.

Characterizing Binding Properties of Protein Interaction Domain

A large proportion of protein-protein interactions are mediated by families of peptide-binding domains. Comprehensive characterization of each of these domains is critical for understanding the mechanisms and networks of protein interaction at the domain level. A systematic experimental strategy was developed for efficient binding property characterization of peptide-binding domains based on high throughput validation screening of a specialized candidate ligand library using yeast two-hybrid system. As for simple adaptor protein without any other known functional domains, the potential functions of the complex were predicted by functional annotations from a MILANO literature search and subcellular localizations. The ligands were considered more likely to be functionally associated if they had similar patterns of functions or closely related functions. For some functionally associated ligand pairs, interaction with one ligand was found to be influenced by another ligand in a yeast three-hybrid system. Ideally protein-protein interactions should be studied with high throughput computational approaches first to screen protein sequence databases to direct the validating experiments toward the most promising peptides. An integrated machine learning systems was built using three novel coding methods and used to screen the Swiss-Prot and GenBank protein databases for potential ligands of SH3 and PDZ domains. A large fraction of predictions has already been experimentally confirmed by other independent research groups, indicating a satisfying generalization capability for future applications in identifying protein interactions. S2/July 2008/Special Issue

Can flow cytometry play a part in cell based high‐content screening?

In parallel to gene therapy and cellular therapy it is clear that the more traditional pharmacological approaches still have a major role to play in the treatment of most diseases. Two drug-discovery strategies are widely used in the pharmaceutical industry: the so-called rational drug design and the most recent screening-based strategy. Rational drug design requires usually an excellent knowledge of the biomolecular target: molecular mechanisms of action, three-dimensional structure, etc. It is scientifically sound and often efficient but rather time consuming. To cope with an exponentially increasing number of potentially interesting targets derived from genomic and proteomic studies, the industry has developed the screening-based approach since the 1990s. It has proven to be very efficient to afford original hit compounds that may open the way to drug development. During the last decades

Rapid characterization of the binding property of HtrA2/Omi PDZ domain by validation screening of PDZ ligand library

HtrA2/Omi is a mammalian mitochondrial serine protease, and was found to have dual roles in mammalian cells, not only acting as an apoptosis-inducing protein but also maintaining mitochondrial homeostasis. PDZ domain is one of the most important protein-protein interaction modules and is involved in a variety of important cellular functions, such as signal transduction, degradation of proteins, and formation of cytoskeleton. Recently, it was reported that the PDZ domain of HtrA2/Omi might regulate proteolytic activity through its interactions with ligand proteins. In this study, we rapidly characterized the binding properties of HtrA2/Omi PDZ domain by validation screening of the PDZ ligand library with yeast two-hybrid approach. Then, we predicted its novel ligand proteins in human proteome and reconfirmed them in the yeast two-hybrid system. Finally, we analyzed the smallest networks bordered by the shortest path length between the protein pairs of novel interactions to evaluate the confidence of the identified interactions. The results revealed some novel binding properties of HtrA2/Omi PDZ domain. Besides the reported Class II PDZ motif, it also binds to Class I and Class III motifs, and exhibits restricted variability at P(-3), which means that the P(-3) residue is selected according to the composition of the last three residues. Seven novel ligand proteins of HtrA2/Omi PDZ domain were discovered, providing significant clues for further clarifying the roles of HtrA2/Omi. Moreover, this study proves the high efficiency and practicability of the newly developed validation screening of candidate ligand library method for binding property characterization of peptide-binding domains.