Alterations in mitochondrial and apoptosis-regulating gene expression in photodynamic therapy-resistant variants of HT29 colon carcinoma cells

Photodynamic therapy (PDT) is a novel cancer therapy inducing irreversible photodamage to tumor tissue via photosensitizer-mediated oxidative cytotoxicity. The cellular and molecular responses associated with PDT are only partially understood. We have reported previously the generation of several photosensitizer-specific PDT-resistant cell variants of HT29 human colon adenocarcinoma cells by selecting cells from sequential PDT treatment using different photosensitizers. In this report, we describe the use of messenger RNA (mRNA) differential display to identify genes that were differentially expressed in the parental HT29 cells compared with their resistant variants. In comparison with parental HT29 cells, mRNA expression was increased in the PDT-resistant cell variants for BNIP3, estrogen receptor-binding fragment-associated gene 9, Myh-1c, cytoplasmic dynein light chain 1, small membrane protein I and differential dependent protein. In contrast, expression in the PDT-resistant variants was downregulated for NNX3, human HepG2 3' region Mbol complementary DNA, glutamate dehydrogenase, hepatoma-derived growth factor and the mitochondrial genes coding for 16S ribosomal RNA (rRNA) and nicotinamide adenine dinucleotide (NADH) dehydrogenase subunit 4. The reduction for mitochondrial 16S rRNA in the PDT-resistant variants was confirmed by Northern blotting, and the elevated expression of the proapoptotic BNIP3 in the PDT-resistant variants was confirmed by Northern and Western blotting analysis. We also examined the expression of some additional apoptosis-regulating genes using Western blotting. We show an increased expression of Bcl-2 and heat shock protein 27 and a downregulation of Bax in the PDT-resistant variants. In addition, the mutant p53 levels in the parental HT29 cells were reduced substantially in the PDT-resistant variants. We suggest that the altered expression in several mitochondrial and apoptosis-regulating genes contributes to PDT resistance.     

Targeting of phage display vectors to mammalian cells

Phage display libraries offer a strategy to isolate peptide ligands to target proteins and to define potential interaction sites between proteins. Recent studies have indicated a novel utility for phage display in that bacteriophage engineered to express peptide ligands to specific cell surface receptors are internalized by mammalian cells. Thus, reporter genes such as green fluorescent protein and lacZ harbored in the phage genome can be delivered to mammalian cells using targeting peptides displayed on the surface of phage. There is also the possibility to generate novel types of peptide libraries expressed intracellularly using a phage capable of inducing expression of its coding genes in human cells.     

Differential display identifies genes in chinese hamster ovary cells sensitive to elevated ammonium

Ammonium is a toxic waste product that has been reported to negatively inhibit cell growth and recombinant glycosylation in Chinese hamster ovary (CHO) cells; however, the effect of this toxicity on intracellular gene expression has received only limited investigation. We used a differential display method to identify genes in CHO cells that were affected by ammonium stress. Eight genes whose mRNA levels significantly changed in response to elevated ammonium were isolated and identified. Five of the genes were identified as having lower expression under the ammonium stress, whereas three genes were identified as having higher expression. Sequence homology with other mammalian organisms was used to attribute function to these newly identified genes. The identified ammonium-sensitive genes were grouped into three broad functional groups: cellular processes, energy metabolism, and genetic-information processing. The three cellular process-related genes had lower expression (anaphase-promoting complex subunit 5, eukaryotic initiation factor 5A II, KIAA1091 protein). The two energy-related genes had higher expression under ammonium stress (adenosine triphosphate synthase subunit C and mitofusin 1). Both of the genetic information-processing genes (endoplasmic reticulum [ER]-resident protein ERdj5 and structure-specific recognition protein 1) had lower expression under the ammonium stress, whereas the 26S proteasome subunit adenosine triphosphatase 3 gene had higher expression. These preliminary results indicate that ammonium stress lowers expression of genes controlling cell cycle, protein folding, and quality and raises genes that control energy metabolism and degradation. Our findings demonstrate the usefulness of mRNA differential-display techniques for the detection of CHO cell genes affected by ammonium stress.     

Differential display competitive polymerase chain reaction: an optimal tool for assaying gene expression

Gene discovery, i.e. detection of genes whose expression is affected in diseases or by different treatments of cells or animals, has become the focus of much genetic research. The technologies that are used to detect changes in expression level include polymerase chain reaction (PCR)-based subtraction methods, arrays of cDNA clones on chips or filters, serial analysis of gene expression, and differential display. In this paper we show that differential display can be used to investigate global gene expression in situations where a few genes change expression levels such as exposure of MCF7 cells to estradiol, and in more complex situations such as neuronal differentiation of human NTERA2 cells which affects a large number of genes. Furthermore, we show that differential display can replace Northern blotting and RNase protection as a tool to study the expression level of a specific gene in many samples. Results obtained by differential display can be stored in databases, where the identity of a band (gene or mRNA name) can be linked with information about the primer combination displaying the band and a gel image showing the band pattern, which is all the information that is needed to compare the expression level of this gene in other samples.     

Insights regarding novel biomarkers and the pathogenesis of primary colorectal carcinoma based on bioinformatic analysis

BackgroundBiomarkers are important in the study of tumor processes for early detection and precise treatment. The biomarkers that have been previously detected are not useful for clinical application for primary colorectal carcinoma (PCRC). The aim of this study was to explore clinically valuable biomarkers of PCRC based on integrated bioinformatic analysis.Material and methodsGene expression data were acquired from the GSE41258 dataset, and the differentially expressed genes were determined between PCRC and normal colorectal samples. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analyses were implemented via Gene Set Enrichment Analysis. A protein-protein interaction (PPI) network was constructed. The significant modules and hub genes were screened and identified in the PPI network.ResultsA total of 202 DEGs were identified, including 58 upregulated and 144 downregulated genes in PCRC samples compared to those in normal colorectal samples. Enrichment analysis demonstrated that the gene sets enriched in PCRC were significantly related to bicarbonate transport, regulation of sodium ion transport, potassium ion homeostasis, regulation of telomere maintenance, and other processes. A total of 10 hub genes was identified by cytoHubba: PYY, CXCL3, CXCL11, CXCL8, CXCL12, CCL20, MMP3, P2RY14, NPY1R, and CXCL1.ConclusionThe hub genes, such as NPY1R, P2RY14, and CXCL12, and the electrolyte disequilibrium resulting from the differential expression of genes, especially bicarbonate imbalance, may provide novel insights and evidence for the future diagnosis and targeted therapy of PCRC.     

Analysis of Differentially Expressed Proteins in Colorectal Cancer Using Hydroxyapatite Column and SDS-PAGE

Limitation on two dimensional (2D) gel electrophoresis technique causes some proteins to be under presented, especially the extreme acidic, basic, or membrane proteins. To overcome the limitation of 2D electrophoresis, an analysis method was developed for identification of differentially expressed proteins in normal and cancerous colonic tissues using self-pack hydroxyapatite (HA) column. Normal and cancerous colon tissues were homogenized and proteins were extracted using sodium phosphate buffer at pH 6.8. Protein concentration was determined and the proteins were loaded unto the HA column. HA column reduced the complexity of proteins mixture by fractionating the proteins according to their ionic strength. Further protein separation was accomplished by a simple and cost effective sodium dodecyl sulfate-polyacrylamide gel electrophoresis method. The protein bands were subjected to in-gel digestion and protein analysis was performed using electrospray ionization (ESI) ion trap mass spectrometer. There were 17 upregulated proteins and seven downregulated proteins detected with significant differential expression. Some of these proteins were low abundant proteins or proteins with extreme pH that were usually under presented in 2D gel analysis. We have identified brain mitochondrial carrier protein 1, T-cell surface glycoprotein CD1a, SOSS complex subunit B2, and Protein Jade 1 which were previously not detected in 2D gel analysis method.     

Enrichment of low abundance proteins of Escherichia coli by hydroxyapatite chromatography.

Visualization of low-copy-number gene products is essential for the detection of novel drug targets by differential protein expression studies. We investigated the enrichment of low-abundance proteins of Escherichia coli by hydroxyapatite chromatography. The proteins of the various pools collected from a ceramic hydroxyapatite column were analyzed by two-dimensional electrophoresis and identified by matrix-assisted laser desorption ionization mass spectrometry. Approximately 800 spots corresponding to 296 different proteins were identified in the hydroxyapatite eluate. About 130 proteins that had not been detected in the two-dimensional gels of the total extract were identified. Hydroxyapatite chromatography enriched low-abundance but also major components of the E. coli protein extract. In particular, it enriched many low-molecular-mass proteins, such as cold-shock proteins. The proteins bound to the hydroxyapatite matrix belong to several classes, including enzymes with various catalytic activities, heat- and cold-shock proteins and many hypothetical and novel proteins with yet unknown functions. The results include a list of the proteins enriched by hydroxyapatite chromatography and a two-dimensional map of the enriched proteins. They may be useful in the design of protein purification pathways using master purification steps and in the search for novel drug targets.     

Large-scale differential display analysis of T helper cell differentiation

We have developed a novel large-scale multicapillary fluorescent differential display (FDD) platform amenable to further automation. The power of the method is demonstrated by the analysis of T helper cell differentiation. Eight RNA samples from wild type, Stat4 knockout and Stat6 knockout mice were analyzed with 16 anchoring primers and 24 arbitrary primers, resulting in 285 294 sample peaks. Visually selected patterns of differential expression suggest two major regulatory mechanisms: activation and Stat4 genotype. A subset of the findings is reproduced in the confirmatory differential display (DD) that included technical and biological replicates. In a small fragment identification pilot study, we identify Ifi27 and Cct8 to be up-regulated by T cell activation. We present a method for the analysis of electropherogram similarity across large datasets, based on correlation of low-resolution representations of electrophoretic data. We show how it can be applied to analyze experimental and technical variables. Using this method, we demonstrate the effect of activation and genotype. In addition, agreement of our real experimental data to the theoretical basis of DD, as well as issues in anchoring primer selectivity, are studied.     

Proteomic study of the peripheral proteins from thylakoid membranes of the cyanobacterium Synechocystis sp. PCC 6803

Thylakoid membranes of cyanobacteria and plants contain enzymes that function in diverse metabolic reactions. Many of these enzymes and regulatory proteins are associated with the membranes as peripheral proteins. To identify these proteins, we separated and identified the peripheral proteins of thylakoid membranes of the cyanobacterium Synechocystis sp. PCC 6803. Trichloroacetic acid (TCA)-acetone extraction was used to enrich samples with peripheral proteins and to remove integral membrane proteins. The proteins were separated by two-dimensional electrophoresis (2-DE) and identified by peptide mass fingerprinting. More than 200 proteins were detected on the sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gel that was stained with colloidal Coomassie blue. We analyzed 116 spots by peptide mass fingerprinting and identified 78 spots that were derived from 51 genes. Some proteins were found in multiple spots, indicating differential modifications resulting in charge differences. Therefore, a significant fraction of the peripheral proteins in thylakoid membranes is modified post-translationally. In our analysis, products of 17 hypothetical genes could be identified in the peripheral protein fraction. Therefore, proteomic analysis is a powerful tool to identify location of the products of hypothetical genes and to characterize complexity in gene expression due to post-translational modifications.     

Identification of gel-separated tumor marker proteins by mass spectrometry

Two-dimensional gel electrophoresis with subsequent analysis by mass spectrometry was applied to study differences in protein expression between benign and malignant solid tumors from human beast, lung and ovary cells. Cells from freshly resected clinical material were lysed and the extracts were subjected to isoelectric focusing with immobilized pH gradients followed by second-dimensional separation on 10-13% sodium dodecyl sulfate (SDS)/polyacrylamide gels. Polypeptides were identified using matrix-assisted laser desorption/ionization and electrospray ionization mass spectrometry after in-gel protein digestion. Some of the upregulated polypeptides in malignant cells are of potential importance as markers of tumor proliferation. Twenty such proteins were identified, ten constituting novel identifications and ten sequence verifications of previously gel-matched proteins. The proteins identified span a wide range of functions, but several cases of protein truncation were found. Truncated forms of cytokeratins 6D and 8, and of cathepsin D were identified. Truncated froms of these over-expressed proteins support the presence of proteolytic processing steps in tumor material. The protein processing and the difference between protein and mRNA abundancies in tumors of different malignancy and origin suggest that studies at the protein level are important for an understanding of tumor phenotypes.     

Evaluation of biological monitoring markers using genomic and proteomic analysis for automobile emission inspectors and waste incinerating workers exposed to polycyclic aromatic hydrocarbons or 2,3,7,8,-tetracholrodedibenzo-p-dioxins

In this study, we investigated the effects of PAHs and dioxin on mRNA and plasma protein expression using genomic and proteomic analysis for automobile emission inspectors and waste incineration workers. About 54 workers from automobile emission inspection offices, 31 workers from waste incinerating company and 84 unexposed healthy subjects were enrolled in this study. Urine and air samples were collected and analyzed by HPLC and GC/MS. Comet assays were carried out to evaluate any DNA damage in mononuclear and polynuclear cells. A significant difference in Olive tail moments in mononuclear cells was observed between exposed and control subjects (P < 0.0001). To examine the differences of the gene expression profile in automobile emission inspectors and waste incineration workers, radioactive complementary DNA microarrays were used to evaluate changes in the expression of 1,152 total genes. The gene expression profiles showed that 11 genes were up-regulated and 4 genes were down-regulated in waste incinerating workers as compared with controls. Plasma proteins were analyzed by 2-dimentional electrophoresis with pH 3-10 NL IPG Dry strip. The protein expression profiles showed that 8 proteins were up- regulated and 1 protein, haptoglobin, was down- regulated in automobile emission inspectors and waste incineration workers. Serum paraoxonase/ arylesterase was found only in the plasma of waste incineration workers. The expression of genes and proteins involved in oxidative stress were up-regulated in both automobile emission inspectors and waste incineration workers. Several proteins, such as transthyrethin, sarcolectin and haptoglobin, that were highly up- or down-regulated, could serve as biological monitoring markers for future study.     

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.     

Novel application of Ag nanoclusters in fluorescent imaging of human serum proteins after native polyacrylamide gel electrophoresis (PAGE)

We have developed a novel application for DNA oligonucleotide-stabilized Ag nanoclusters in fluorescent imaging of human serum proteins after native polyacrylamide gel electrophoresis (PAGE). Oligonucleotide-stabilized Ag nanoclusters were used as fluorescent probes for direct detection of proteins after native PAGE. Some relatively low-abundance proteins, such as α-1-antichymotrypsin (ACT) and α-2-glycoprotein 1, zinc (ZAG) were easily detected by oligonucleotide-stabilized Ag nanocluster-based fluorescent imaging and identified by MS and MS/MS techniques, without the need of expensive antibodies or tedious immunoassay procedures. The pH condition for the oligonucleotide-stabilized Ag nanocluster solution was optimized and the possible mechanism of interaction between proteins and DNA oligonucleotide-stabilized Ag nanoclusters was analyzed. As a novel fluorescent detection method it is simple, fast, nontoxic and sensitive, and it shows great analytical potential in proteome research and in biochemistry.     

Protein abundance alterations in matched sets of macroscopically normal colon mucosa and colorectal carcinoma

Our current results, aimed at the detection of protein abundance alterations that could be associated with the process of colon tumorigenesis, are summarized. The matched sets of macroscopically normal colon mucosa and colorectal carcinoma were examined by a one- or two-dimensional electrophoretic approach and proteins were identified using immunoblotting or mass spectrometry. The following results were observed: The levels of liver fatty acid-binding protein, actin-binding protein/smooth muscle protein 22-alpha and cyclooxygenase 2 were downregulated in colorectal carcinoma compared to normal colon mucosa. Conversely, the expression of a novel variant of heat shock protein70 and several members of the S100 protein family of calcium-binding proteins (two isoforms of S100A9, S100A8, S100A11 and S100A6) were upregulated in transformed colon mucosa. Despite the variations of the levels of expression of given protein among analyzed samples, all quantitative changes were found to be statistically significant (Mann-Whitney test assuming p < or = 0.05). We conclude that the proteomic approach is useful for the study of complex biological events underlying the process of colorectal tumorigenesis.