Biomedical subjects

Xianqiang Li

Publications and source records attributed to Xianqiang Li.

9 recordsLinked to original sources

PubMed2006

Src homology 2 domain-based high throughput assays for profiling downstream molecules in receptor tyrosine kinase pathways.

Src homology 2 (SH2) domains are evolutionary conserved small protein modules that bind specifically to tyrosine-phosphorylated peptides. More than 100 SH2 domains have been identified in proteins encoded by the human genome. The binding specificity of these domains plays a critical role in signaling within the cell, mediating the relocalization and interaction of proteins in response to changes in tyrosine phosphorylation states. Here we developed an SH2 domain profiling method based on a multiplexed fluorescent microsphere assay in which various SH2 domains are used to probe the global state of tyrosine phosphorylation within a cell and to screen synthetic peptides that specifically bind to each SH2 domain. The multiplexed, fluorescent microsphere-based assay is a recently developed technology that can potentially detect a wide variety of interactions between biological molecules. We constructed 25-plex SH2 domain-GST fusion protein-conjugated fluorescent microsphere sets to investigate phosphorylation-mediated cell signaling through the specific binding of SH2 domains to activated target proteins. The response of HeLa, COS-1, A431, and 293 cells and four breast cancer cell lines to epidermal growth factor and insulin were quantitatively profiled using this novel microsphere-based, multiplexed, high throughput assay system.

Amino Acid Sequence↗

PubMed2006

Development of a fluorescent microsphere-based multiplexed high-throughput assay system for profiling of transcription factor activation.

Transcription factors (TFs), which play crucial roles in the regulation of gene expression in the human genome, are highly regulated by a variety of mechanisms. A single extracellular stimulus can trigger multiple signaling pathways, and these in turn can activate multiple TFs to mediate the inducible expression of target genes. Alterations in the activities of TFs are often associated with human diseases, such as altered activating factor 1, estrogen receptor, and p53 function in cancer, nuclear factor kappaB in inflammatory diseases, and peroxisome proliferator-activated receptor gamma in obesity. A systematic assay for profiling the activation of TFs will aid in elucidating the mechanisms of TF activation, reveal altered TFs associated with human diseases, and aid in developing assays for drug discovery. Here, we developed a 24-plex fluorescent microsphere-based TF activation assay system with a 96-well plate format. The assay system enabled high-throughput profiling of the DNA binding activity of TFs in multiple samples with high sensitivity.

DNA↗

PubMed2006

Profiling activities of transcription factors in breast cancer cell lines.

Transcription factors (TFs) are critical regulators of cell growth and differentiation, whose dysfunction is associated with many human diseases, including cancer. To facilitate the discovery of functionally altered TFs among the approximately 2,000 human TFs, we (Panomics, Inc., Fremont, CA) developed a Protein/DNA array technology that can be used to profile the activities of multiple TFs simultaneously. In this study, we applied this technology to examine the TF activities in three different breast cancer cell lines: MCF7 (estrogen receptor [ER] +, tamoxifen-sensitive), T47D (ER+, tamoxifen-resistant), and HCC1806 (ER-, tamoxifen-resistant). We compared the differences in TF activity in these cells lines following treatment with estradiol or tamoxifen. We found a number of TF activities unique to each of these cell lines. In addition to verifying previous findings, the novel findings of this study provide a more comprehensive view of the differences in the response of these cancer lines to estrogen and tamoxifen.

Animals↗

PubMed2006

Development of luciferase reporter-based cell assays.

Using luciferase reporter constructs driven by specific promoter response elements, we developed a series of stable reporter cell lines for monitoring the activity of specific transcription factors (TFs). These TFs, which play essential roles in regulating diverse biological functions, include nuclear factor kappaB (NFkappaB), cyclic AMP response element-binding protein, activator protein 1, signal transducer and activator of transcription 1 and 3, nuclear factor of activated T cells, serum response factor, and hypoxia-inducible factor. The response of the stable reporter cells was highly specific. For example, tumor necrosis factor-alpha (TNFalpha) strongly activated NFkappaB reporter cells, but not other cell lines. The NFkappaB reporter was active in multiple cell lines, including 293T, HeLa, A549, and NIH3T3 cells, in response to TNFalpha, indicating that this system is useful to monitor specific TFs in different model cell lines. To facilitate high throughput screening of these cell lines, they were adapted to a 96-well format. These stable reporter cells are also applicable for the analysis of steroid hormone receptors, which bind directly to the response element after ligand binding. With the HeLa/glucocorticoid response element-luciferase stable reporter cells, we were able to discriminate pharmacological activity of different compounds for the glucocorticoid receptor. Taken together, these results demonstrate that the stable reporter cells are useful tools for: (1) detection of signaling pathway-specific ligands; (2) identification of novel ligands for specific TFs, and (3) screening for agonists and antagonists of specific ligands/receptors.

Animals↗

PubMed2006

High throughput assays for analyzing transcription factors.

Transcription factors are a group of proteins that modulate the expression of genes involved in many biological processes, such as cell growth and differentiation. Alterations in transcription factor function are associated with many human diseases, and therefore these proteins are attractive potential drug targets. A key issue in the development of such therapeutics is the generation of effective tools that can be used for high throughput discovery of the critical transcription factors involved in human diseases, and the measurement of their activities in a variety of disease or compound-treated samples. Here, a number of innovative arrays and 96-well format assays for profiling and measuring the activities of transcription factors will be discussed.

Biological Assay↗

PubMed2004

Development of a method for screening short-lived proteins using green fluorescent protein.

We have developed a screening technology for the identification of short-lived proteins. A green fluorescent protein (GFP)-fusion cDNA library was generated for monitoring degradation kinetics. Cells expressing a subset of the GFP-cDNA expression library were screened to recover those in which the fluorescence signal diminished rapidly when protein synthesis was inhibited. Thirty clones that met the screening criteria were characterized individually. Twenty-three (73%) proved to have a half-life of 4 hours or less.

Cell Line↗

PubMed2004

Protein-DNA array-based identification of transcription factor activities regulated by interaction with the glucocorticoid receptor.

The glucocorticoid receptor (GR) regulates gene expression by binding specific sequence elements within the promoters of target genes or by cross-talk with other transcription factors (TFs). For some TFs, interaction with the GR results in alteration of DNA binding and transcriptional regulation. We used a protein-DNA array, a system that facilitates simultaneous profiling of the activities of multiple transcription factors, to systematically examine the potential cross-talk of GRalpha with 149 TFs. Using this array, we identified several TFs, including IRF, E47, and COUP-TF, whose DNA binding activities were modulated by GRalpha. We then confirmed these results with in vitro electrophoretic mobility shift assays and in vivo reporter assays. In this study, IRF and E47 were identified as participants in GRalpha cross-talk for the first time. This new finding expands our understanding of the functional role of GRalpha in the context of gene expression regulation.

Actins↗

PubMed2004

SH3 domain protein-binding arrays.

First identified as part of the Rous sarcoma oncogene product Src, SH3 (Src Homology 3) domains play an important role in intercellular communication and intracellular signal transduction. A high-throughput assay for ligand binding to SH3 domains--SH3 domain proteins immobilized on a membrane--allows rapid visualization of numerous SH3 domain protein-protein interactions with no expensive equipment or radioactivity required. Once the array is constructed or obtained commercially, the procedure is straightforward: The protein of interest is cloned into a fusion-tagged expression vector and expressed in bacteria, the prepared bacterial extract is incubated with the array membrane, and the signal is measured using a chemiluminescence detection system.

Humans↗

PubMed2003

Use of an array technology for profiling and comparing transcription factors activated by TNFalpha and PMA in HeLa cells.

Multiple signal transduction pathways are generally triggered simultaneously by a single extracellular stimulus. As a result, multiple transcription factors (TFs) can be activated downstream to mediate the inducible expression of target genes. Profiling the activation of all TFs will aid in the dissection of the numerous pathways of signal transduction. Tumor necrosis factor alpha (TNFalpha) and phorbol 12-myristate 13-acetate (PMA) mediate many biological functions, including cell proliferation and apoptosis, by stimulating signaling pathways. Two TFs, nuclear factor kappaB (NFkappaB) and activating factor 1 (AP1), have been identified as targets of both TNFalpha and PMA activation. Here, we describe the use of a protein/DNA array system to identify additional TFs activated by TNFalpha and PMA in HeLa cells. From a total of 150 targeted TFs, six-CREB, E2F, CETP/CRE, c-Rel, MSP1, and Pax6-were identified whose activities, like NFkappaB and AP1, were regulated by both TNFalpha- and PMA-induced pathways. Interestingly, the TF E47 was shown to be specifically activated by TNFalpha but was not affected by treatment with PMA. In addition, GATA, NF-E1, and ISRE were shown to be specifically activated by PMA but not TNFalpha. These findings suggest that TNFalpha and PMA both stimulate unique signaling pathways while mediating transcriptional activation through common pathways.

DNA Fingerprinting↗