A STRATEGY FOR ISOLATING DIFFERENTIATIONINDUCING COMPLEMENTARY DNAs FROM HUMAN ESOPHAGEAL CANCER CELL LINE TREATED WITH RETINOIC ACID

Treatment of the human esophageal cancer cell line EC8712 with retinoic acid (RA) stopped the cell growth significantly and gave rise to terminal differentiation of the cells characterized by increased expression of involucrin gene. Two cDNA libraries were constructed from the parental and RA-treated cells respectively. Repeated subtractive hybridization of single-stranded plasmid DNA prepared from pooled colonies of cDNA library of the parental cells with cDNA probe generated from the RA-treated cells exhausted sequences common to both libraries of the cell. The unhybridized cDNA probe represented, therefore, the genes activated after RA-treatment. By using these enriched cDNAs as probe to screen the cDNA library constructed from the RA-treated cells thirty-nine positive colonies were obtained, of which two were specifically due to RA-induction. One of these two cDNA clones, designated as pRA538, has undergone further analysis and shown differentiation-inducing effect on parental cancer cells. A novel     

A strategy for isolating differentiation-inducing complementary DNAs from human esophageal cancer cell line treated with retinoic acid.

Treatment of the human esophageal cancer cell line EC8712 with retinoic acid (RA) stopped the cell growth significantly and gave rise to terminal differentiation of the cells characterized by increased expression of involucrin gene. Two cDNA libraries were constructed from the parental and RA-treated cells respectively. Repeated subtractive hybridization of single-stranded plasmid DNA prepared from pooled colonies of cDNA library of the parental cells with cDNA probe generated from the RA-treated cells exhausted sequences common to both libraries of the cell. The unhybridized cDNA probe represented, therefore, the genes activated after RA-treatment. By using these enriched cDNAs as probe to screen the cDNA library constructed from the RA-treated cells thirty-nine positive colonies were obtained, of which two were specifically due to RA-induction. One of these two cDNA clones, designated as pRA538, has undergone further analysis and shown differentiation-inducing effect on parental cancer cells. A novel strategy for cloning genes involved in terminal differentiation of cancer cells is developed.     

The cloning of human genes using cDNA phage display and small-molecule chemical probes

The cloning of genes based on protein function has become a powerful tool for protein discovery and should play an important role in proteomics in general. We have recently reported a technique for the functional identification of protein targets by combining traditional affinity chromatography with cDNA phage display. This procedure, referred to as display cloning, directly couples biologically active natural products to the gene of their protein cellular target. We now report the cloning of a human gene, the domain of F1 ATP synthase, using a synthetic scaffold molecule which serves as a prototype for a diverse chemical library. The ability to select genes from cDNA libraries using probes from combinatorial libraries would greatly increase the number of small molecule/protein interactions that can be identified. This method might prove valuable in furthering our understanding of biology and its application toward drug development.     

Display cloning: functional identification of natural product receptors using cDNA-phage display.

BACKGROUND: The identification of cellular targets has traditionally been the starting point for natural product mode of action studies and has led to the understanding of many biological processes. Conventional experimental approaches have depended on cell-based screening and/or affinity chromatography. Although both of these techniques aid in the discovery of protein cellular targets, a method that couples protein identification with gene isolation would be extremely valuable. RESULTS: A procedure for the direct cloning of cellular proteins, based on their affinity for natural products, using cDNA phage display has been developed. The technique is referred to as display cloning because it involves the cloning of proteins displayed on the surface of a bacteriophage particle. The approach has been established by isolating a full-length gene clone of FKBP12 (FK506-binding protein) from a human brain cDNA library using a biotinylated FK506 probe molecule. During the affinity selection, the FKBP12 gene emerged as the dominant library member and was the only sequence identified after the second round of selection. CONCLUSIONS: The development of display cloning greatly facilitates the investigation of ligand-receptor interaction biology and natural product mode of action studies. This procedure utilizes heterologous protein display on infectious phage, which allows the amplification and repeated selection of putative sequences, leading to unambiguous target identification. In addition, the direct connection of a functional protein to its gene sequence eliminates the subsequent cloning step required with tissue homogenate or cell lysate affinity methods, allowing direct isolation of an expressible gene sequence.     

Mapping the high throughput SEREX technology screening for novel tumor antigens

Advances in novel tumor-associated antigen (TAA) screening strategy have accelerated the identification and characterization of biomarkers and potential target molecules for tumor subtyping, diagnosis and therapeutics, which may facilitate early detection and diagnosis of the diseases individually and enhance treatment approaches for cancer. Over the past decades, a plethora of non-invasive methodologies dedicated to identify novel target molecules have been primarily focusing on the discovery of human tumor antigens recognized by the autologous antibody repertoire or cytotoxic T lymphocytes, among which serological analysis of recombinant cDNA expression libraries (SEREX) technology is chronologically first established and is of outstanding sensitivity and antigen coverage. This approach involves immunoscreening cDNA libraries extracted from fresh tumor tissues with sera from cancer patients to identify gene products recognized by IgG antibody. SEREX-defined clones can be directly sequenced and their expression profiles can be readily determined, allowing for immediate structural definition of the antigenic target and subsequent identification of TAAs and their cognate autoantibodies. This review is not only devoted to outline the SEREX technology and its advantages, drawbacks and recent modifications currently available for discovering provocative tumor antigens, but also to translate these SEREX-defined peptides into valuable cancer-specific signatures that would aid in the development of diagnostics, prognostics and therapeutics for cancer patients.     

The powerful combination of phage surface display of cDNA libraries and high throughput screening

Phage surface display of cDNA libraries facilitates cloning, expression and rapid selection of functional gene products physically linked to their genetic information through gene product-ligand interactions. Efficient screening technologies based on selective enrichment of clones expressing desired gene products allows, within a short time, the isolation of all ligand-specific clones that are present in a library. Manual identification of clones by restriction analysis and random sequencing is unlike to be successful for the isolation of gene products derived from rare mRNA species resulting from selection of the libraries using polyvalent ligands like serum from patients. Here we describe rapid handling of large numbers of individual clones selected from molecular libraries displayed on phage surface using the power of robotics-based high throughput screening. The potential of the combination of cDNA-phage surface display, with selection for specific interactions by functional screening and robotic technology is illustrated by the isolation of more sequences potentially encoding IgE-binding proteins than postulated from Western blot analyses using extracts derived from raw material of complex allergenic sources. The subsequent application of functional enrichment and robotics-based screening will facilitate the rapid generation of information about the repertoire of protein structures involved in allergic diseases.     

20 years of DNA-encoded chemical libraries

The identification of specific binding molecules is a central problem in chemistry, biology and medicine. Therefore, technologies, which facilitate ligand discovery, may substantially contribute to a better understanding of biological processes and to drug discovery. DNA-encoded chemical libraries represent a new inexpensive tool for the fast and efficient identification of ligands to target proteins of choice. Such libraries consist of collections of organic molecules, covalently linked to a unique DNA tag serving as an amplifiable identification bar code. DNA-encoding enables the in vitro selection of ligands by affinity capture at sub-picomolar concentrations on virtually any target protein of interest, in analogy to established selection methodologies like antibody phage display. Multiple strategies have been investigated by several academic and industrial laboratories for the construction of DNA-encoded chemical libraries comprising up to millions of DNA-encoded compounds. The implementation of next generation high-throughput sequencing enabled the rapid identification of binding molecules from DNA-encoded libraries of unprecedented size. This article reviews the development of DNA-encoded library technology and its evolution into a novel drug discovery tool, commenting on challenges, perspectives and opportunities for the different experimental approaches.     

Study of the difference of high and low metastasis cell line's gene expression map and metastasis-related genes of adenoid cystic carcinoma

We searched for metastasis-related genes in adenoid cystic carcinoma by suppression subtractive hybridization analysis of high and low metastasis cell lines. Twelve genes (ten previously identified and two novel sequences) were identified as being expressed at lower levels in high metastasis cell line Acc-M when compared to low metastasis cell line Acc-2. The known sequences corresponded to the genes for cysteine-rich angiogenesis induction factor (cyr61), chromosome 7 RP11-52501 clone, G-protein, WAS familial ferritin I heavy chain, jumping translocation breakpoint, eukaryotic translation elongation, folate receptor and three ribosomal proteins. Among them, the G protein and ferritin I heavy chain genes contained mutations in the high metastasis cell line. The two novel gene sequences have been named ACC metastasis-associated RNH and ACC metastasis-associated suspected protein (GenBank # AF522024 and AF522025, respectively). Taken together, these results suggest that reduced expression and/or mutation of several genes in the tumor cell line Acc-M are associated with high tumor metastasis, providing important molecular biological materials for further study of metastasis control and possible targets for cancer gene therapy.     

Forward chemical genetics: library scaffold design

With the unraveling of the entire human genome, it has become imperative to understand the function of the gene products, proteins. Within the past several years, chemical genetics has gained recognition as a powerful approach to study protein function by using small molecules as gene knock-out or knock-in mimics. Forward chemical genetics is a three-step process; the design and synthesis of a small molecule library represents the first step followed secondly by the search for novel phenotypes and then by isolation and identification of target protein(s). This review will focus on the first step, the design of the scaffold for small molecule libraries. It will also examine the connection between the choice of a scaffold and the propensity of that library to demonstrate enhanced biological activity when tested in certain cellular systems.     

High-throughput screening of surface displayed gene products

With the human genome project approaching completion, there is a growing interest in functional analysis of gene products. The characterization of large numbers of proteins, their expression patterns and in vivo localisations, demands the use of automated technology that maintains a logistic link to the encoding genes. As a complementary approach, phage display is used for recombinant protein expression and the selection of interacting (binding) molecules. Cloning of libraries in filamentous bacteriophage or phage mid vectors provides a physical link between the expressed protein and its encoding DNA sequence. High-throughput technology for automated library handling and phage display selection has been developed using picking-spotting robots and a module for pin-based magnetic particle handling. This system enables simultaneous interaction screening of libraries and the selection of binders to different target molecules at high throughput. Target molecules are either displayed on high-density filter membranes (protein filters) or tag-bound to magnetic particles and can be handled as native ligands. Binding activity is confirmed by magnetic particle ELISA in the microtitre format. The whole procedure from immobilisation of target molecules to confirmed clones of binders is automatable. Using this technology, we have selected human scFv antibody fragments against expression products of human cDNA libraries.     

Design and screening of M13 phage display cDNA libraries

The last decade has seen a steady increase in screening of cDNA expression product libraries displayed on the surface of filamentous bacteriophage. At the same time, the range of applications extended from the identification of novel allergens over disease markers to protein-protein interaction studies. However, the generation and selection of cDNA phage display libraries is subjected to intrinsic biological limitations due to their complex nature and heterogeneity, as well as technical difficulties regarding protein presentation on the phage surface. Here, we review the latest developments in this field, discuss a number of strategies and improvements anticipated to overcome these challenges making cDNA and open reading frame (ORF) libraries more readily accessible for phage display. Furthermore, future trends combining phage display with next generation sequencing (NGS) will be presented.     

Gene Expression in Secondary Metabolism and Metabolic Switching Phase of <Emphasis Type="Italic">Phanerochaete chrysosporium</Emphasis>

Ligninolytic enzymes are well-known to play the crucial roles in lignin biodegradation and have potential applications in industrial processes. The filamentous white-rot fungus, Phanerochaete chrysosporium, has been widely used as a model organism for studying these ligninolytic enzymes that are able to degrade the lignin during the secondary metabolism. To study the gene expression in secondary metabolism and metabolic switching phase of P. chrysosporium, we constructed a metabolic-switching phase suppression subtractive hybridization (SSH) cDNA library and a secondary metabolic phase SSH cDNA library to compare their mRNA expression profiles. We isolated the genes that are specially expressed and subsequently identified four genes that specially expressed during metabolic-switching phase while 22 genes in secondary metabolic phase. Accordingly, these specially expressed genes might play key roles in different metabolic stages, which would offer more new insights into the shift from nitrogen to lignin metabolism.     

Phage display of combinatorial peptide libraries: application to antiviral research

Given the growing number of diseases caused by emerging or endemic viruses, original strategies are urgently required: (1) for the identification of new drugs active against new viruses and (2) to deal with viral mutants in which resistance to existing antiviral molecules has been selected. In this context, antiviral peptides constitute a promising area for disease prevention and treatment. The identification and development of these inhibitory peptides require the high-throughput screening of combinatorial libraries. Phage-display is a powerful technique for selecting unique molecules with selective affinity for a specific target from highly diverse combinatorial libraries. In the last 15 years, the use of this technique for antiviral purposes and for the isolation of candidate inhibitory peptides in drug discovery has been explored. We present here a review of the use of phage display in antiviral research and drug discovery, with a discussion of optimized strategies combining the strong screening potential of this technique with complementary rational approaches for identification of the best target. By combining such approaches, it should be possible to maximize the selection of molecules with strong antiviral potential.     

Expressed Sequence Tag Analysis of the Dinoflagellate Lingulodinium polyedrum During Dark Phase¶

To collect information on gene expression during the dark period in the luminous dinoflagellate Lingulodinium polyedrum, normalized complementary DNA (cDNA) libraries were constructed from cells collected during the first hour of night phase in a 12:12 h light‐dark cycle. A total of 4324 5′‐end sequence tags were isolated. The sequences were grouped into 2111 independent expressed sequence tags (EST) from which 433 groups were established by similarity searches of the public nonredundant protein database. Homology analysis of the total sequences indicated that the luminous dinoflagellate is more similar to land plants and animals (vertebrates and invertebrates) than to prokaryotes or algae. We also isolated three bioluminescence‐related (luciferase and two luciferinbinding proteins [LBP]) and 37 photosynthesis‐related genes. Interestingly, two kinds of LBP genes occur in multiple copies in the genome, in contrast to the single luciferase gene. These cDNA clones and EST sequence data should provide a powerful resource for future genome‐wide functional analyses for uncharacterized genes.     

Privileged structures: applications in drug discovery

Over the past 15 years the privileged structure concept has emerged as a fruitful approach to the discovery of novel biologically active molecules. Privileged structures are molecular scaffolds with versatile binding properties, such that a single scaffold is able to provide potent and selective ligands for a range of different biological targets through modification of functional groups. In addition, privileged structures typically exhibit good drug-like properties, which in turn leads to more drug-like compound libraries and leads. The net result is the production of high quality leads that provide a solid foundation for further development. The identification of privileged structures will be discussed, emphasizing the importance of understanding the structure-target relationships that confer "privileged" status. This understanding allows privileged structure based libraries to be targeted at distinct target families (e.g. GPCRs, LGIC, enzymes/kinases). Privileged structures have been successfully exploited across and within different target families and promises to be an effective approach to the discovery and optimization of novel bioactive molecules. The application of the privileged structure approach, both in traditional medicinal chemistry and in the design of focused libraries, will be discussed with the aid of illustrative examples.