[Protein chip, an innovative high throughput method for detection of functional proteins].
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Biotechnology↗
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Results for “Protein Array Analysis”
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Distinct roles of activating transcription factor 6 (ATF6) and double-stranded RNA-activated protein kinase-like endoplasmic reticulum kinase (PERK) in transcription during the mammalian unfolded protein response.
In response to accumulation of unfolded proteins in the endoplasmic reticulum (ER), a homoeostatic response, termed the unfolded protein response (UPR), is activated in all eukaryotic cells. The UPR involves only transcriptional regulation in yeast, and approx. 6% of all yeast genes, encoding not only proteins to augment the folding capacity in the ER, but also proteins working at various stages of secretion, are induced by ER stress [Travers, Patil, Wodicka, Lockhart, Weissman and Walter (2000) Cell (Cambridge, Mass.) 101, 249-258]. In the present study, we conducted microarray analysis of HeLa cells, although our analysis covered only a small fraction of the human genome. A great majority of human ER stress-inducible genes (approx. 1% of 1800 genes examined) were classified into two groups. One group consisted of genes encoding ER-resident molecular chaperones and folding enzymes, and these genes were directly regulated by the ER-membrane-bound transcription factor activating transcription factor (ATF) 6. The ER-membrane-bound protein kinase double-stranded RNA-activated protein kinase-like ER kinase (PERK)-mediated signalling pathway appeared to be responsible for induction of the remaining genes, which are not involved in secretion, but may be important after cellular recovery from ER stress. In higher eukaryotes, the PERK-mediated translational-attenuation system is known to operate in concert with the transcriptional-induction system. Thus we propose that mammalian cells have evolved a strategy to cope with ER stress different from that of yeast cells.
Activating Transcription Factor 6↗
Gene expression profiles of light-induced apoptosis in arrestin/rhodopsin kinase-deficient mouse retinas.
To examine the molecular processes that lead to light-induced retinal degeneration, mutant mice deficient in arrestin and rhodopsin kinase were raised in the dark and then subjected to relatively low doses of white light. The kinetics of the subsequent induction of apoptosis, change in mRNA transcript level, and photoreceptor cell death were monitored. Analysis of transcript profiles identified clusters of genes that responded differently to illumination, including a cluster of photoreceptor-specific genes that showed marked decreases in levels long before morphological damage could be readily ascertained. The behaviors of other gene clusters demonstrate the coordinate induction of stress gene responses early in the course of irradiation. There was little, if any, change in transcript levels corresponding to genes associated with the initiation of apoptosis or antiapoptotic effects. Transcript analysis provides insight into the patterns of gene expression that are associated with the different stages of retinal degeneration in this model system.
Animals↗
Mass spectrometry meets chip technology: a new proteomic tool in cancer research?
DNA chip technologies are the most exiting genomic tools, which were developed within the last few years. It is, however, evident that knowledge of the gene sequence or the quantity of gene expression is not sufficient to predict the biological nature and function of a protein. This can be particularly important in cancer research where post-translational modifications of a protein can specifically contribute to the disease. To address this problem, several proteomic tools have been developed. Currently the most widely used proteomic tool is two-dimensional protein gel electrophoresis (2-DE), which can display protein expression patterns to a high degree of resolution. As an alternative to 2-DE, a preliminary study using a new technique was employed to generate protein expression patterns from whole tissue extracts. Surface-enhanced laser desorption/ionization (SELDI) allows the retention of proteins on a solid-phase chromatographic surface (ProteinChip Array) with direct detection of retained proteins by time of flight-mass spectrometry (TOF-MS). Using this system, we analyzed eight cases of renal cell carcinoma (RCC) including normal, peripheral and central tumor tissue as well as four microdissected cases of cervical intraepithelial neoplasia (CIN) and three microdissected cases of cervix uteri carcinoma. Differentially expressed proteins were found by comparing the protein expression patterns generated using SELDI-based TOF-MS of tumor tissue with normal and neoplastic tissue, respectively. By applying this fast and powerful ProteinChip array technology it becomes possible to investigate complex changes at the protein level in cancer associated with tumor development and progression.
Carcinoma, Renal Cell↗
Identification and characterization of rapidly dividing U937 clones with differential telomerase activity and gene expression profiles: role of c-Myc/Mad1 and Id/Ets proteins.
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Apoptosis↗
Influence of the period-dependent circadian clock on diurnal, circadian, and aperiodic gene expression in Drosophila melanogaster.
We measured daily gene expression in heads of control and period mutant Drosophila by using oligonucleotide microarrays. In control flies, 72 genes showed diurnal rhythms in light-dark cycles; 22 of these also oscillated in free-running conditions. The period gene significantly influenced the expression levels of over 600 nonoscillating transcripts. Expression levels of several hundred genes also differed significantly between control flies kept in light-dark versus constant darkness but differed minimally between per(01) flies kept in the same two conditions. Thus, the period-dependent circadian clock regulates only a limited set of rhythmically expressed transcripts. Unexpectedly, period regulates basal and light-regulated gene expression to a very broad extent.
Animals↗
Dose-dependent and independent temporal patterns of gene responses to ionizing radiation in normal and tumor cells and tumor xenografts.
U87 cells derived from human malignant gliomas and growtharrested human embryonic lung (HEL) fibroblasts were examined with respect to their response to ionizing radiation by profiling their RNAs. In the first series of experiments, cells grown in vitro were harvested and the RNAs were extracted 5 h after exposure to 1, 3, or 10 Gy. In the second series of experiments the U87 tumors were implanted in nude mice and subjected to the same doses of irradiation. The xenografts were harvested at 1, 5, or 24 h after irradiation and subjected to the same analyses. We observed and report on (i) cell-type common and cell-type specific responses, (ii) genes induced at low levels of irradiation but not at higher doses, (iii) temporal patterns of gene response in U87 xenografts that varied depending on radiation dose and temporal patterns of response that were similar at all doses tested, (iv) significantly higher up-regulation of cells in xenografts than in in vitro cultures, and (v) genes highly up-regulated by radiation. The responding genes could be grouped into nine functional clusters. The representation of the nine clusters was to some extent dependent on dose and time after irradiation. The results suggest that clinical outcome of ionizing radiation treatment may benefit significantly by taking into account both cell-type and radiation-dose specificity of cellular responses.
Animals↗
Differentially expressed genes in asbestos-induced tumorigenic human bronchial epithelial cells: implication for mechanism.
Although exposure to asbestos fibers is associated with the development of lung cancer, the underlying mechanism(s) remains unclear. Using human papillomavirus-immortalized human bronchial epithelial (BEP2D) cells, we previously showed that UICC chrysotiles can malignantly transform these cells in a stepwise fashion before they become tumorigenic in nude mice. In the present study we used cDNA expression arrays to screen differentially expressed genes among the tumorigenic cells. A total of 15 genes were identified, 11 of which were further confirmed by northern blot. Expression levels of these genes were then determined among transformed BEP2D cells at different stages of the neoplastic process, including non-tumorigenic cells that were resistant to serum-induced terminal differentiation, early and late passage transformed BEP2D cells, five representative tumor cell lines and fused tumorigenic-control cell lines which were no longer tumorigenic. A consistent 2- to 3-fold down-regulation of the DCC (deleted in colon cancer), Ku70 and heat shock protein 27 genes were detected in all the independently generated tumor cell lines while expression levels in early transformants as well as in the fusion cell lines remained normal. In contrast, all the tumor cell lines examined demonstrated 2- to 4-fold overexpression of the insulin receptor and its signal transduction genes. Differential expression of these genes was completely restored in the fusion cell lines examined. No alteration in c-jun or EGF receptor expression was found in any of the cell lines. Our data suggest that activation of the insulin receptor pathway and inactivation of DCC and Ku70 may cooperate in malignant transformation of BEP2D cells induced by asbestos.
Animals↗
The Toll and Imd pathways are the major regulators of the immune response in Drosophila.
Microarray studies have shown recently that microbial infection leads to extensive changes in the Drosophila gene expression programme. However, little is known about the control of most of the fly immune-responsive genes, except for the antimicrobial peptide (AMP)-encoding genes, which are regulated by the Toll and Imd pathways. Here, we used oligonucleotide microarrays to monitor the effect of mutations affecting the Toll and Imd pathways on the expression programme induced by septic injury in Drosophila adults. We found that the Toll and Imd cascades control the majority of the genes regulated by microbial infection in addition to AMP genes and are involved in nearly all known Drosophila innate immune reactions. However, we identified some genes controlled by septic injury that are not affected in double mutant flies where both Toll and Imd pathways are defective, suggesting that other unidentified signalling cascades are activated by infection. Interestingly, we observed that some Drosophila immune-responsive genes are located in gene clusters, which often are transcriptionally co-regulated.
Animals↗
Identification of genes encoding mouse oocyte secretory and transmembrane proteins by a signal sequence trap.
The oocyte plays a key role in follicular development. At all stages of follicular development, oocytes interact with surrounding granulosa cells and promote their differentiation into the types of cells that support further oocyte growth and developmental competence. These interactions suggest the existence of an oocyte-granulosa cell regulatory loop that includes both secreted proteins and cell surface receptors on both cell types. Factors involved in the regulatory loop will therefore contain a signal sequence, which can be used to identify them through a signal sequence trap (SST). A screen of an oocyte SST library identified three classes of oocyte-expressed sequences: known mouse genes, sequences homologous to known mammalian genes, and novel sequences of unknown function. Many of the recovered genes may have roles in the oocyte-granulosa cell regulatory loop. For several of the known mouse genes, new roles in follicular development are implied by identification of their expression, for the first time, in the oocyte. The future characterization of novel sequences may lead to the identification of novel proteins participating in the regulatory loop.
Animals↗
Identification of NF-kappaB-dependent gene networks in respiratory syncytial virus-infected cells.
Respiratory syncytial virus (RSV) is a mucosa-restricted virus that is a leading cause of epidemic respiratory tract infections in children. In epithelial cells, RSV replication activates nuclear translocation of the inducible transcription factor nuclear factor kappaB (NF-kappaB) through proteolysis of its cytoplasmic inhibitor, IkappaB. In spite of a putative role in mediating virus-inducible gene expression, the spectrum of NF-kappaB-dependent genes induced by RSV infection has not yet been determined. To address this, we developed a tightly regulated cell system expressing a nondegradable, epitope-tagged IkappaBalpha isoform (Flag-IkappaBalpha Mut) whose expression could be controlled by exogenous addition of nontoxic concentrations of doxycycline. Flag-IkappaBalpha Mut expression potently inhibited IkappaBalpha proteolysis, NF-kappaB binding, and NF-kappaB-dependent gene transcription in cells stimulated with the prototypical NF-kappaB-activating cytokine tumor necrosis factor alpha (TNF-alpha) and in response to RSV infection. High-density oligonucleotide microarrays were then used to profile constitutive and RSV-induced gene expression in the absence or presence of Flag-IkappaBalpha Mut. Comparison of these profiles revealed 380 genes whose expression was significantly changed by the dominant-negative NF-kappaB. Of these, 236 genes were constitutive (not RSV regulated), and surprisingly, only 144 genes were RSV regulated, representing numerically approximately 10% of the total population of RSV-inducible genes at this time point. Hierarchical clustering of the 144 RSV- and Flag-IkappaBalpha Mut-regulated genes identified two discrete gene clusters. The first group had high constitutive expression, and its expression levels fell in response to RSV infection. In this group, constitutive mRNA expression was increased by Flag-IkappaBalpha Mut expression, and the RSV-induced decrease in expression was partly inhibited. In the second group, constitutive expression was very low (or undetectable) and, after RSV infection, expression levels strongly increased. In this group, NF-kappaB was required for RSV-inducible expression because Flag-IkappaBalpha Mut expression blocked their induction by RSV. This latter cluster includes chemokines, transcriptional regulators, intracellular proteins regulating translation and proteolysis, and secreted proteins (complement components and growth factor regulators). These data suggest that NF-kappaB action induces global cellular responses after viral infection.
Anti-Bacterial Agents↗
Inactivation of DNA mismatch repair by increased expression of yeast MLH1.
Inactivation of DNA mismatch repair by mutation or by transcriptional silencing of the MLH1 gene results in genome instability and cancer predisposition. We recently found (P. V. Shcherbakova and T. A. Kunkel, Mol. Cell. Biol. 19:3177-3183, 1999) that an elevated spontaneous mutation rate can also result from increased expression of yeast MLH1. Here we investigate the mechanism of this mutator effect. Hybridization of poly(A)(+) mRNA to DNA microarrays containing 96.4% of yeast open reading frames revealed that MLH1 overexpression did not induce changes in expression of other genes involved in DNA replication or repair. MLH1 overexpression strongly enhanced spontaneous mutagenesis in yeast strains with defects in the 3'-->5' exonuclease activity of replicative DNA polymerases delta and epsilon but did not enhance the mutation rate in strains with deletions of MSH2, MLH1, or PMS1. This suggests that overexpression of MLH1 inactivates mismatch repair of replication errors. Overexpression of the PMS1 gene alone caused a moderate increase in the mutation rate and strongly suppressed the mutator effect caused by MLH1 overexpression. The mutator effect was also reduced by a missense mutation in the MLH1 gene that disrupted Mlh1p-Pms1p interaction. Analytical ultracentrifugation experiments showed that purified Mlh1p forms a homodimer in solution, albeit with a K(d) of 3.14 microM, 36-fold higher than that for Mlh1p-Pms1p heterodimerization. These observations suggest that the mismatch repair defect in cells overexpressing MLH1 results from an imbalance in the levels of Mlh1p and Pms1p and that this imbalance might lead to formation of nonfunctional mismatch repair complexes containing Mlh1p homodimers.
Adaptor Proteins, Signal Transducing↗
Gene expression screening of human mast cells and eosinophils using high-density oligonucleotide probe arrays: abundant expression of major basic protein in mast cells.
Mast cells (MCs) and eosinophils are thought to play important roles in evoking allergic inflammation. Cell-type--specific gene expression was screened among 12,000 genes in human MCs and eosinophils with the use of high-density oligonucleotide probe arrays. In comparison with other leukocytes, MCs expressed 140 cell-type--specific transcripts, whereas eosinophils expressed only 34. Among the transcripts for expected MC-specific proteins such as tryptase, major basic protein (MBP), which had been thought to be eosinophil specific, was ranked fourth in terms of amounts of increased MC-specific messenger RNA. Mature eosinophils were almost lacking this transcript. MCs obtained from 4 different sources (ie, lung, skin, adult peripheral blood progenitor--derived and cord blood progenitor--derived MCs, and eosinophils) were found to have high protein levels of MBP in their granules with the use of flow cytometric and confocal laser scanning microscopic analyses. The present finding that MCs can produce abundant MBP is crucial because many reports regarding allergic pathogenesis have been based on earlier findings that MBP was almost unique to eosinophils and not produced by MCs. (Blood. 2001;98:1127-1134)
Adult↗
CD30-activation-mediated growth inhibition of anaplastic large-cell lymphoma cell lines: apoptosis or cell-cycle arrest?
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Antibodies, Monoclonal↗
Small proline-rich repeat protein 1A is expressed by axotomized neurons and promotes axonal outgrowth.
The ability of neurons to regenerate an axon after injury is determined by both the surrounding environment and factors intrinsic to the damaged neuron. We have used cDNA microarrays to survey those genes induced during successful sciatic nerve regeneration. The small proline-rich repeat protein 1A (SPRR1A) is not detectable in uninjured neurons but is induced by >60-fold after peripheral axonal damage. The protein is localized to injured neurons and axons. sprr1a is one of a group of epithelial differentiation genes, including s100c and p21/waf, that are coinduced in neurons by axotomy. Overexpressed SPRR1A colocalizes with F-actin in membrane ruffles and augments axonal outgrowth on a range of substrates. In axotomized sensory neurons, reduction of SPRR1A function restricts axonal outgrowth. Neuronal SPRR1A may be a significant contributor to successful nerve regeneration.
Actins↗
Coordinate gene expression patterns during osteoblast maturation and retinoic acid treatment of MC3T3-E1 cells.
The clonal osteoblast-like cell line MC3T3-E1 undergoes time-dependent morphological changes leading to the formation of bone nodules in vitro. Serial analysis of gene expression was used to identify genes expressed in MC3T3-E1 cells, including known osteoblast markers, structural and matrix proteins, transcription factors, cell-cycle regulators, and housekeeping genes. Relative changes in the expression of 92 representative transcripts were determined by arrayed cDNA hybridization. Complex probes were derived from MC3T3-E1 cells during the proliferation, differentiation, and matrix mineralization stages, and from cells grown with all- trans-retinoic acid (RA), a potent bone morphogen. We found that the relative expression of 68 of these genes was higher during differentiation than in the earlier proliferative phase. In the mineralization phase, all but 16 cDNAs had lower normalized hybridization intensity ratios as compared with the differentiation phase. cDNAs for vimentin (Vim), presenilin 2 (Psen2), guanine nucleotide binding protein alpha stimulating (Gnas), gap-junction membrane channel protein beta 1 (Gjb1), fibroblast growth factor receptor 1 (Fgfr1), eukaryotic translation elongation factor 2 (Eef2), and calponin 2 (Cnn2) had higher normalized hybridization intensities in both differentiation and mineralization than in proliferation. RA treatment during the differentiation phase appeared to reduce the expression of the 92 genes examined, as 62 cDNAs had lower hybridization intensities with complex cDNA probes derived from RA-treated cells than with the probe from untreated cells. Several cDNAs representing genes with previously unrecognized RA responsiveness were identified by this comparison, including the receptor for bone morphogenetic proteins 2 and 4 (Bmp2/Bmp4) and hemoglobin Y.
Activin Receptors, Type I↗
Dissecting the circuitry of protein kinase A and cAMP signaling in cancer genesis: antisense, microarray, gene overexpression, and transcription factor decoy.
Expression of the RI alpha subunit of the cAMP-dependent protein kinase type I (PKA-I) is enhanced in human cancer cell lines, in primary tumors, in transformed cells, and in cells upon stimulation of growth. Signaling via the cAMP pathway may be complex, and the biological effects of the pathway in normal cells may depend upon the physiological state of the cells. However, results of different experimental approaches such as antisense exposure, 8-Cl-cAMP treatment, and gene overexpression have shown that the inhibition of RI alpha/PKA-I exerts antitumor activity in a wide variety of tumor-derived cell lines examined in vitro and in vivo. cDNA microarrays have further shown that in a sequence-specific manner, RI alpha antisense induces alterations in the gene expression profile of cancer cells and tumors. The cluster of genes that define the "proliferation-transformation" signature are down-regulated, and those that define the "differentiation-reverse transformation" signature are up-regulated in antisense-treated cancer cells and tumors, but not in host livers, exhibiting the molecular portrait of the reverted (flat) phenotype of tumor cells. These results reveal a remarkable cellular regulation, elicited by the antisense RI alpha, superimposed on the regulation arising from the Watson-Crick base-pairing mechanism of action. Importantly, the blockade of both the PKA and PKC signaling pathways achieved with the CRE-transcription factor decoy inhibits tumor cell growth without harming normal cell growth. Thus, a complex circuitry of cAMP signaling comprises cAMP growth regulatory function, and deregulation of the effector molecule by this circuitry may underlie cancer genesis and tumor progression.
8-Bromo Cyclic Adenosine Monophosphate↗
Identification of differentially expressed genes in esophageal squamous cell carcinoma (ESCC) by cDNA expression array: overexpression of Fra-1, Neogenin, Id-1, and CDC25B genes in ESCC.
PURPOSE: This study aims to identify differentially expressed genes in esophageal squamous cell carcinoma (ESCC) through the use of a membrane-based cDNA array. EXPERIMENTAL DESIGN: Two newly established human ESCC cell lines (HKESC-1 and HKESC-2) and one corresponding to a morphologically normal, esophageal epithelium tissue specimen, prospectively collected from the HKESC-2-related patient, were screened in parallel using a cDNA expression array containing gene-specific fragments for 588 human genes spotted onto nylon membranes. RESULTS: The results of cDNA expression array showed that 53 genes were up-regulated 2-fold or higher and 8 genes were down-regulated 2-fold or higher in both ESCC cell lines at the mRNA level. Semiquantitative RT-PCR analysis of a subset of these differentially expressed genes gave results consistent with cDNA array findings. Four of the differentially expressed genes that belong to the categories of oncogenes/tumor suppressor genes (Fra-1 and Neogenin) and cell cycle-related genes (Id-1 and CDC25B) were studied more extensively for their protein expression by immunohistochemistry. The two ESCC cell lines and their corresponding primary tissues, 61 primary ESCC resected specimens and 16 matching, morphologically normal, esophageal epithelium tissues were analyzed. The immunostaining results showed that Fra-1, Neogenin, Id-1, and CDC25B were overexpressed in both ESCC cell lines and their corresponding primary tumors at the protein level, validating the microarray findings. The results of the clinical specimens showed that the Fra-1 gene was overexpressed in ESCC compared with normal esophageal epithelium in 53 of 61 cases (87%), Neogenin in 57 of 61 cases (93%), Id-1 in 57 of 61 cases (93%), and CDC25B in 48 of 61 cases (79%). Furthermore, the expression of Fra-1, Neogenin, and Id-1 in ESCC correlated with tumor differentiation. CONCLUSIONS: Overall, this study demonstrates that multiple genes are differentially expressed in ESCC and provides the first evidence that oncogenes Fra-1 and Neogenin and cell cycle-related genes Id-1 and CDC25B are overexpressed in ESCC.
Carcinoma, Squamous Cell↗