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PubMed2003

Functional proteomic analysis of melanoma progression.

Functional proteomics provides a powerful approach to screen for alterations in protein expression and posttranslational modifications under conditions of human disease. In this study, we use protein screening to examine markers of melanoma progression, by profiling melanocyte versus melanoma cell lines using two-dimensional electrophoresis and mass spectrometry. Eight candidate markers were identified as differentially regulated in transformed cells. In particular, hepatoma-derived growth factor (HDGF) and nucleophosmin B23 were strongly correlated with melanoma. Nucleophosmin B23 is a nucleolar and centrosome-associated protein, which has been implicated as a target for cyclin E/cyclin-dependent kinase 2 (CDK2) in modulating centrosome duplication and cell cycle control. Western blotting of one-dimensional and two-dimensional gels showed that the form of nucleophosmin B23 that is up-regulated in melanoma represents a posttranslationally modified form, most likely reflecting enhanced phosphorylation in the tumor-derived cells. In contrast, Western analysis of HDGF demonstrated increased expression of all forms in melanoma cell lines compared with melanocytes. Immunohistochemical analysis of human tissue biopsies showed strong expression of HDGF in early and late stage melanomas and low expression in melanocytes and nontumorigenic nevi. Interestingly, biopsies of nevi showed a graded effect in which HDGF immunoreactivity was reduced in nevoid nests penetrating deep into the dermis compared with nests at the epidermal-dermal junction, suggesting that HDGF expression in nevi is dependent on epidermal cell interactions. In contrast, biopsies of melanoma showed strong expression of HDGF throughout the tumor, including cells located deeply within dermis. Thus, expression of this antigen likely reports a reduced dependence of protein expression on epidermal interactions.

Biomarkers, Tumor↗

PubMedDate not supplied

Peptidomic and proteomic analyses of the systemic immune response of Drosophila.

Insects have developed an efficient host defense against microorganisms, which involves humoral and cellular mechanisms. Numerous data highlight similarities between defense responses of insects and innate immunity of mammals. The fruit fly, Drosophila melanogaster, is a favorable model system for the analysis of the first line defense against microorganisms. Taking advantages of improvements in mass spectrometry (MS), two-dimensional (2D) gel electrophoresis and bioinformatics, differential analyses of blood content (hemolymph) from immune-challenged versus control Drosophila were performed. Two strategies were developed: (i) peptidomic analyses through matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) MS and high performance liquid chromatography for molecules below 15 kDa, and (ii) proteomic studies based on 2D gel electrophoresis, MALDI-TOF fingerprinting and database searches, for compounds of greater molecular masses. The peptidomic strategy led to the detection of a large number of peptides induced in the hemolymph of challenged flies as compared to controls. Of these, 28 were characterized, amongst which were antimicrobial peptides. The 2D gel electrophoresis strategy led to the detection of 70 spots differentially regulated by at least fivefold after microbial infection. This approach yielded the identity of a series of proteins that were related to the Drosophila immune response, such as proteases, protease inhibitors, prophenoloxydase-activating enzymes, serpins and a Gram-negative binding protein-like protein. This strategy also brought to light new candidates with a potential function in the immune response (odorant-binding protein, peptidylglycine alpha-hydroxylating monooxygenase and transferrin). Interestingly, several molecules resulting from the cleavage of proteins were detected after a fungal infection. Together, peptidomic and proteomic analyses represent new tools to characterize molecules involved in the innate immune reactions of Drosophila.

Animals↗

PubMed2006

[The effects of angiotensin II on gene expression of hepatic stellate cells].

OBJECTIVE: Our previous investigation demonstrated that angiotensin II could induce proliferation and differentiation of hepatic stellate cells, and also could up-regulate its extracellular matrix synthesis. The objective of this study was to determine the effects of 10(-5) mol/L angiotensin II on gene expression of hepatic stellate cells. METHODS: After incubation with 10(-5) mol/L angiotensin II for 48 hours, the cultured hepatic stellate cells were collected. The mRNA and total protein were obtained from cell lysate and then suppression subtractive hybridization (SSH), 2D-gel electrophoresis and MALDI-TOF mass spectrometry were used to identify these cDNAs and proteins. RESULTS: A total of 36 clones from the subtracted cDNA library were sequenced and compared to sequences in the GenBank using BLAST. Of the 36 differentially expressed gene fragments from the subtracted library, 13 differentially expressed gene fragments showed significant homology to other known proteins, such as ribosomal protein, beta-actin FE-3, leucyl-tRNA synthetase, CD147, pyruvate kinase, peroxiredoxin 1, and BAT3, while 2 other gene fragments encoding protein BC097361 and BC057380 and their functions were not disclosed. About 1110 and 1008 protein spots were detected by employing the ImageMaster 2D Platinum 4.9 proteome image analysis system in angiotensin-treated hepatic stellate cells and control cells separately. Among these spots 108 proteins were up-regulated while the other 153 proteins were down-regulated. Several up-regulated proteins were chosen to be excised and in-gel digest MALDI-TOF-MS and Database analysis showed that among the high expression proteins, there were prohibitin, RBL-NDP kinase 1.8x10(4) subunit, electron transfer flavoprotein alpha-subunit, guanine nucleotide binding protein, alpha 15, and heat shock 7.0x10(4) protein 5. CONCLUSION: Our results suggest that the up-regulation of hepatic stellate cell mRNA influences proliferation, differentiation and apoptosis of those cells. The proteins of signal transduction, metabolizing regulation, apoptosis suppression, and fibrogenesis regulation of hepatic stellate cells were up-regulated.

Angiotensin II↗

PubMed2000

[Transcriptome analysis and pharmacogenomics].

Pharmacogenomics is defined as identification of loci which are involved in determining the responsiveness and distinguishing responders and non-responders to a given drug. Genome sequencing, transcriptome and proteome analysis are of particular significance in pharmacogenomics. Sequencing is used to locate polymorphisms, and monitoring of gene expression can provide clues about the genomic response to disease and treatment. The transcriptome analysis can be done by methods of random cDNA sequencing (expressed sequence tag project, body map project, serial analysis of gene expression, etc.), mRNA display (differential display, fluorescent differential display, RNA arbitrary primed PCR, molecular indexing, gene expression fingerprinting, etc.) and differential hybridization (cDNA high density filter, cDNA microarray, oligomicrochip, etc.). We describe the principle and application of trancriptome analysis in pharmacogenomics, especially differential display and cDNA microarray. We used transcriptome analysis to identify therapeutic target genes by studying the change of gene expression in animal models of oxidative stress and hypoxia and found novel drug target candidates through this pharmacogenomic strategy.

Animals↗

PubMed2025

Multi-omics signature of healthy versus unhealthy lifestyles reveals associations with diseases.

This multi-omics cross-sectional study investigated differences in metabolomics, proteomics, and epigenomics profiles between two groups of adults matched for age but differing in lifestyle factors such as body composition, diet, and physical activity patterns. Data from prior studies were utilized for a comprehensive integrative analysis. The study included 52 participants in the lifestyle group (LIFE) (28 males, 24 females) and 52 in the control group (CON) (27 males, 25 females). Using multi-omics integration software (OmicsNet and Pathview), 96 significantly (p < 0.05) enriched pathways were identified that differentiated the LIFE and CON groups. Top pathways significantly (p < 2.63 × 10-5) influenced by group status included fatty acid degradation, fatty acid elongation, glutathione metabolism, Parkinson disease, and central carbon metabolism in cancer. This study identified a distinct metabolic signature comprised of metabolites, proteins, and gene methylation sites associated with a healthy lifestyle. These findings provide unique, but complementary, results to previous single-omics analyses using metabolomics and proteomics procedures which showed that the LIFE group exhibited lower plasma bile acid levels, higher levels of beneficial fatty acids, reduced innate immune activation, enhanced lipoprotein metabolism, and increased HDL remodeling. The current multi-omics analysis builds on these previous results by providing a more holistic view of how metabolites, proteins, and methylation sites associated with a healthy lifestyle, providing a larger, more comprehensive list of altered pathways. Additionally, the integrated analysis revealed connections between lifestyle factors and conditions such as cancer and insulin resistance beyond what identified in the single-omics approaches, highlighting the broader metabolic impact of lifestyle on health. Overall, the signatures identified by this multi-omics approach provide a basis for developing more translational biomarkers, such as those that defined the cancer and insulin resistance pathways that can be used to assess one's state of health and provide guidance on behavior modifications that should be taken to lower disease risk.

Humans↗

PubMed2004

Protein expression patterns in primary carcinoma of the vagina.

Protein patterns in six samples from primary vaginal cancers, in five from normal vaginal tissue and in five primary cervical cancers, were analysed using two-dimensional polyacrylamide gel electrophoresis (2-DE). Protein expression profile was evaluated by computer-assisted image analysis (PDQUEST) and proteins were subsequently identified using matrix-assisted laser desorption/ionisation mass spectrometry. The aim was to analyse the protein expression profiles using the hierarchical clustering method in vaginal carcinoma and to compare them with the protein pattern in cervical carcinoma in order to find a helpful tool for correct classification and for increased biomedical knowledge. Protein expression data of a distinct set of 33 protein spots were differentially expressed. These differences were statistically significant (Mann-Whitney signed-Ranked Test, P<0.05) between normal tissue, vaginal and cervical cancer. Furthermore, protein profiles of pairs of primary vaginal and cervical cancers were found to be very similar. Some of the protein spots that have so far been identified include Tropomyosin 1, cytokeratin 5, 15 and 17, Apolipoprotein A1, Annexin V, Glutathione-S-transferase. Others are the stress-related proteins, calreticulin, HSP 27 and HSP 70. We conclude that cluster analysis of proteomics data allows accurate discrimination between normal vaginal mucosa, primary vaginal and primary cervical cancer. However, vaginal and cervical carcinomas also appear to be relatively homogeneous in their gene expression, indicating similar carcinogenic pathways. There might, further, be a possibility to identify tumour-specific markers among the proteins that are differentially expressed. The results from this study have to be confirmed by more comprehensive studies in the future.

Adenocarcinoma↗

PubMed2006

A hormone and proteome approach to picturing the initial metabolic events during Plasmodiophora brassicae infection on Arabidopsis.

We report on the early response of Arabidopsis thaliana to the obligate biotrophic pathogen Plasmodiophora brassicae at the hormone and proteome level. Using a CYCB1;1::GUS construct, the re-initiation of infection-related cell division is shown from 4 days after inoculation on. Sensitivity to cytokinins and auxins as well as the endogenous hormone levels are evaluated. Both an enhanced cytokinin gene response and an accumulation of isopentenyl adenine and adenosine precede this re-initiation of cell division, whereas an enhanced auxin gene response is observed from 6 days after inoculation on. The alhl mutant, impaired in the cross talk between ethylene and auxins, is resistant to P. brassicae. A differential protein analysis of infected versus noninfected roots and hypocotyls was performed using two-dimensional gel electrophoresis and quantitative image analysis, coupled to matrix-assisted laser desorption ionization time of flight-time of flight mass spectrometry-based protein identification. Of the visualized proteins, 12% show altered abundance compared with the noninfected plants, including proteins involved in metabolism, cell defense, cell differentiation, and detoxification. Combining the hormone and proteome data, we postulate that, at the very first stages of Plasmodiophora infection, plasmodial-produced cytokinins trigger a local re-initiation of cell division in the root cortex. Consequently, a de novo meristematic area is established that acts as a sink for host-derived indole-3-acetic acid, carbohydrates, nitrogen, and energy to maintain the pathogen and to trigger gall development.

Arabidopsis↗

PubMed2004

Proteome analysis of human mesothelial cells during epithelial to mesenchymal transitions induced by shed menstrual effluent.

Peritoneal endometriosis is the result of ectopic implantation and growth of endometrium tissue that has been regurgitated into the abdominal cavity during menstruation. We have previously shown that menstrual effluent induces epithelial to mesenchymal transitions (EMT) in mesothelial cells, which results in cell retraction and exposure of submesothelial extracellular matrix. Since endometrial tissue preferentially adheres to the extracellular matrix, adhesion of endometrial tissue to the peritoneum is facilitated. The EMT were shown to be associated with differential expression and phosphorylation of mesothelial proteins. Using radiolabeling and proteomics we detected changes in protein expression and phosphorylation that occur in mesothelial cells during the EMT process. The identity of 73 proteins, which were obtained from 324 analyzed spots, was confirmed. The expression of 35 proteins involved in organization of the cytoskeleton, signal transduction, regulation of the redox state, and production of ATP, was altered during the EMT process. Four of the identified proteins were differentially phosphorylated: annexin-1, an actin-binding protein and a substrate for receptor tyrosine kinases; tropomyosin-alpha, a regulator of actin filament stability and cell shape; elongation factor 1 delta; ATP synthase beta-chain. In conclusion, factors from menstrual effluent induce specific changes in the expression and phosphorylation status of structural, regulatory and metabolic proteins relevant to the complex process of EMT in mesothelial cells.

Cells, Cultured↗

PubMed2001

Genome evolution at the genus level: comparison of three complete genomes of hyperthermophilic archaea.

We have compared three complete genomes of closely related hyperthermophilic species of Archaea belonging to the Pyrococcus genus: Pyrococcus abyssi, Pyrococcus horikoshii, and Pyrococcus furiosus. At the genomic level, the comparison reveals a differential conservation among four regions of the Pyrococcus chromosomes correlated with the location of genetic elements mediating DNA reorganization. This discloses the relative contribution of the major mechanisms that promote genomic plasticity in these Archaea, namely rearrangements linked to the replication terminus, insertion sequence-mediated recombinations, and DNA integration within tRNA genes. The combination of these mechanisms leads to a high level of genomic plasticity in these hyperthermophilic Archaea, at least comparable to the plasticity observed between closely related bacteria. At the proteomic level, the comparison of the three Pyrococcus species sheds light on specific selection pressures acting both on their coding capacities and evolutionary rates. Indeed, thanks to two independent methods, the "reciprocal best hits" approach and a new distance ratio analysis, we detect the false orthology relationships within the Pyrococcus lineage. This reveals a high amount of differential gains and losses of genes since the divergence of the three closely related species. The resulting polymorphism is probably linked to an adaptation of these free-living organisms to differential environmental constraints. As a corollary, we delineate the set of orthologous genes shared by the three species, that is, the genes that may characterize the Pyrococcus genus. In this conserved core, the amino acid substitution rate is equal between P. abyssi and P. horikoshii for most of their shared proteins, even for fast-evolving ones. In contrast, strong discrepancies exist among the substitution rates observed in P. furiosus relative to the two other species, which is in disagreement with the molecular clock hypothesis.

Archaeal Proteins↗

PubMed2004

New antigenic candidates in multiple sclerosis: identification by serological proteome analysis.

Myelin antigen targets that are clearly associated with pathogenic events in multiple sclerosis (M.S.) patients remain to be defined. We recently demonstrated that the analysis of global IgG antibody response against human brain antigens using one-dimensional (1-D) immunoblotting, allowed us to discriminate M.S. patients from controls (both healthy subjects and patients with Sjögren's syndrome). Additionally, this approach also differentiated the three clinical forms of M.S. Indeed, 42 brain antigenic bands (26 from healthy brain and 16 from the M.S. brain) showed the discriminant IgG immune responses. The aim of our study was to characterize the 26 discriminant antigenic bands detected in healthy brain. Protein identification was successively performed by 1-D and two-dimensional (2-D) immunoblottings using sera from 18 M.S. patients, followed by mass spectrometry (MS) analysis and a database search. One hundred and two antigenic spots were then detected on 2-D immunoblots, with M.S. sera against healthy brains. Sixty-four spots were successfully matched with 2-D Coomassie brillant blue stained gels, which were further selected for MS analysis and annotated leading to the identification of 14 of the 26 discriminant antigens. Thus, serological proteome analysis may provide a useful tool for the identification of potentially new M.S.-associated antigens, whose relevance to physiopathological events remains to be defined.

Adult↗

PubMed2025

A widespread protein misfolding mechanism is differentially rescued by chaperones based on gene essentiality.

Protein misfolding involving changes in non-covalent lasso entanglement (NCLE) status has been proposed based on simulations and biochemical assays of a small number of proteins. Here, we detect hallmarks of these misfolded states across hundreds of proteins by integrating E. coli proteome-wide limited-proteolysis mass spectrometry with structural datasets of protein native structures. Proteins containing native NCLEs are twice as likely to misfold, predominantly in regions where these NCLEs naturally occur. Surprisingly, the chaperones DnaK and GroEL do not typically correct this misfolding, except in the case of essential proteins. Statistical analysis links this differential rescue activity to weaker loop-closing contacts in the NCLEs of essential proteins, suggesting misfolding involving these loops is easier to rectify by chaperones. Molecular simulations indicate a mechanism where premature NCLE loop closure, prior to proper placement of the threading segment, leads to persistent misfolded states. This mechanism explains why, in the mass spectrometry data, proteins with NCLEs are more likely to misfold and misfold in NCLE regions. These results suggest widespread NCLE misfolding, that such misfolded states in non-essential proteins can bypass the refolding action of chaperones, and that some protein sequences may have evolved to allow chaperone rescue from this class of misfolding.

Journal Article↗

PubMed2006

Proteomic analysis of the genotoxicant methylazoxymethanol (MAM)-induced changes in the developing cerebellum.

The genotoxicant methylazoxymethanol (MAM) is a widely used developmental neurotoxin, and its glucoside is an etiological factor for western Pacific amyotrophic lateral sclerosis-parkinsonism-dementia complex (ALS/PDC). Identification of global protein expression changes that occur in response to MAM in the developing cerebellum could provide valuable insight into the potential mechanisms involved in the neurodegeneration process. We have utilized fluorescence 2-dimensional differential gel electrophoresis (2D-DIGE), to determine the protein expression changes that occur during normal cerebellar development and in response to MAM. Three day-old postnatal C57BL/6 mice (PND3) received a single injection of MAM, and the cerebella of postnatal day 4 (PND4) and day 22 (PND22) were analyzed. Approximately, 1400 unique spots were matched and quantified in all samples. Comparison of PND4 and PND22 developing cerebellum showed that a significant fraction of the proteome (approximately 68%) changes at this stage. The immediate response of the developing cerebellum to MAM was minimal (approximately 10%). However, significant differences (27%) were noted 14 days after MAM exposure. In contrast, the transcriptome changes were more pronounced at 24 h compared to 14 days. MAM targeted several proteins networks including transport (e.g., alpha-synuclein), cytoskeletal (e.g., beta-tubulin, vimentin), and mitochondrial (e.g., Atp5b) proteins. Immunochemistry confirmed several of the changes in protein expression (alpha-synuclein). Comparison with gene expression changes revealed that the temporal changes observed in the transcriptome and proteome are not correlative. These studies demonstrate for the first time the potential networks involved during neuronal development and neurodegenerative processes that are perturbed by MAM.

Animals↗

PubMed2005

Proteomic analysis of the expression of proteins related to rice quality during caryopsis development and the effect of high temperature on expression.

Proteins are essential to rice caryopsis development and quality formation. High temperature is an important environmental factor, which may decrease grain quality. In the present study rice caryopsis proteins were profiled by two-dimensional polyacrylamide gel electrophoresis, and differentially expressed proteins were analyzed by liquid chromatography/tandem mass spectrometry. Expressions of more than 400 polypeptide spots during caryopsis development, in response to temperature treatments or between varieties were monitored. Among them, more than 70 differentially expressed polypeptides were analyzed by liquid chromatography/tandem mass spectrometry. We identified 54 proteins with known functions. Of these, 21 were involved with carbohydrate metabolism, 14 with protein synthesis and sorting, and 9 with stress responses. Waxy (Wx) proteins and glutelins were the most significant spots, which increased significantly during development. Allergen-like proteins, PPDK and NADH-SDH, also were expressed during development, implying their physiological roles in caryopsis. Expression of large isoforms of Wx proteins was correlated with the amylose content of rice caryopses. One protein with high GC content in its DNA sequence was correlated with the chalky trait of kernels. High temperature (35/30 degrees C) decreased the expression of Wx proteins, allergen-like proteins, and elongation factor 1beta, but increased the expression of small heat shock proteins (sHSP), glyceraldehyde-3-phosphate dehydrogenase, and prolamin. sHSP was positively correlated with the appearance of chalky kernels. During development, glutelins were phosphorylated and glycosylated, indicating that these molecules were post-translationally modified. Possible functions of the expression of candidate proteins on the grain quality are discussed.

Chromatography, Liquid↗

PubMed2026

Immune Regulatory Signatures Associated with Different Recovery Durations of Delayed Graft Function after Kidney Transplantation.

Delayed graft function (DGF) is a common early complication of kidney transplantation characterized by immune activation. The duration of DGF may significantly affect long-term graft survival, yet the immune mechanisms underlying the different DGF durations remain unclear. Using a functional definition of delayed graft function (fDGF), defined as a failure of serum creatinine to decrease by at least 10% per day for three consecutive days within the first postoperative week, patients were stratified into short-term DGF (SDGF) and long-term DGF (LDGF) groups according to recovery periods. In this exploratory study, targeted proteomic analysis indicated that proteins enriched in SDGF were primarily involved in innate immune responses and acute inflammatory processes, including neutrophil chemotaxis and migration, whereas LDGF exhibited features related to adaptive immune responses and chronic inflammation, such as T-cell differentiation and activation. IL-7 and CCL20 were identified as candidate molecules potentially associated with different DGF durations. Targeted metabolomics revealed disturbances in amino acid metabolism, particularly alanine, aspartate, and glutamate metabolism, as well as in energy metabolism, including the tricarboxylic acid (TCA) cycle, which may be involved in LDGF. These findings provide preliminary insights into immune metabolic features associated with different DGF recovery durations.

Humans↗

PubMed2001

Toward a high-throughput approach to quantitative proteomic analysis: expression-dependent protein identification by mass spectrometry.

The isotope-coded affinity tag (ICAT) technology enables the concurrent identification and comparative quantitative analysis of proteins present in biological samples such as cell and tissue extracts and biological fluids by mass spectrometry. The initial implementation of this technology was based on microcapillary chromatography coupled on-line with electrospray ionization tandem mass spectrometry. This implementation lacked the ability to select proteins for identification based on their relative abundance and therefore to focus on differentially expressed proteins. In order to improve the sample throughput of this technology, we have developed a two-step approach that is focused on those proteins for which the abundance changes between samples: First, a new software program for the automated quantification of ICAT reagent labeled peptides analyzed by microcapillary electrospray ionization time-of-flight mass spectrometry determines those peptides that differ in their abundance and second, these peptides are identified by tandem mass spectrometry using an electrospray quadrupole time-of flight mass spectrometer and sequence database searching. Results from the application of this approach to the analysis of differentially expressed proteins secreted from nontumorigenic human prostate epithelial cells and metastatic cancerous human prostate epithelial cells are shown.

Autoanalysis↗

PubMed2026

Identification of Immune Response-Related Proteomic Biomarkers in Moyamoya Disease Using Serum Olink Proteomics.

Moyamoya disease, a rare chronic cerebrovascular disorder, requires invasive digital subtraction angiography (DSA) for diagnosis. This study employed high-throughput proteomics to identify plasma biomarkers for Moyamoya disease diagnosis. We conducted immunopanel analysis using the Olink platform to evaluate 92 immune-related proteins in plasma samples from 88 Moyamoya disease patients and 88 healthy controls. Key proteins were identified through differential expression analysis, GO, and KEGG enrichment analysis. A diagnostic model was constructed using LASSO regression, Boruta algorithm, and machine learning models including random forest and XGBoost. Validation of these proteins was performed using GEO external data sets, followed by prediction of potential therapeutic drugs and molecular docking validation through pharmacogenomic databases. A total of 44 differentially expressed proteins were identified through the Olink immunopanel, with 12 downregulated and 32 upregulated. GO and KEGG analyses revealed significant enrichment of these proteins in innate immune responses and signaling pathways such as NF-kB and MAPK. Through LASSO, random forest, and protein under-area analysis, four potential biomarkers for Moyamoya disease (MGMT, SIT1, PRDX1, TRAF2) were identified. A diagnostic model using these proteins showed the highest AUC value with the XGBoost model. Additionally, TRAF2 and PRDX1 exhibited significant expression differences in Moyamoya disease patients within the GEO data set. Our study revealed the immune landscape of Moyamoya disease, identified four biomarkers, and established a variety of diagnostic models.

Humans↗

PubMed2001

Proteomic profiling of the cancer microenvironment by antibody arrays.

Critical changes in protein expression that enable tumors to initiate and progress originate in the local tissue microenvironment, and there are increasing indications that these microenvironmental alterations in protein expression play critical roles in shaping and directing this process. As a model to better understand how patterns of protein expression shape the tissue microenvironment, we analyzed protein expression in tissue derived from squamous cell carcinoma of the oral cavity through an antibody microarray approach for high-throughput proteomic analysis. Utilizing laser capture microdissection to procure total protein from specific microscopic cellular populations, we demonstrate that quantitative, and potentially qualitative, differences in expression patterns of multiple proteins within epithelial cells reproducibly correlate with oral cavity tumor progression. Furthermore, differential expression of multiple proteins was also found in stromal cells surrounding and adjacent to regions of diseased epithelium that directly correlated with tumor progression of the epithelium. Most of the proteins identified in both cell types are involved in signal transduction pathways, thus we hypothesize that extensive molecular communication involving complex cellular signaling between epithelium and stroma play a key role in driving oral cavity cancer progression.

Antibodies, Neoplasm↗

PubMed2002

Comparison of protein expression profiles between monolayer and spheroid cell culture of HT-29 cells revealed fragmentation of CK18 in three-dimensional cell culture.

The use of three-dimensional cell culture models, so-called multicellular tumor spheroids, is a special approach in experimental cancer research, because spheroids are similar to in vivo tumors in structural as well as functional sense. Cells grown in spheroids exhibit alterations of cell cycle regulation, induction of apoptosis and differentiation and can acquire multidrug resistance. In this study we investigated the protein expression in human colorectal cancer cells grown in monolayer and in spheroid cultures using proteomics. Evaluation by computer-assisted image analysis revealed overexpression of three cytokeratin 18 fragments that were generated in vivo. Cytokeratin 18 has previously been described as a target for caspase-mediated cleavage during apoptosis and our results indicate that apoptosis may take place in spheroids. Other proteins upregulated in spheroids include calreticulin precursor, a rho GDP dissociation inhibitor variant, several cytokeratins and peroxiredoxin 4. Some of these proteins have already been linked to chemoresistance and apoptotic phenomena.

Electrophoresis, Gel, Two-Dimensional↗