Biomedical subjects

Vladimir Knezevic

Publications and source records attributed to Vladimir Knezevic.

16 recordsLinked to original sources

PubMed2006

Transfer and multiplex immunoblotting of a paraffin embedded tissue.

As we transition from genomics to the challenges of the functional proteome, new tools to explore the expression of proteins within tissue are essential. We have developed a method of transferring proteins from a formalin fixed, paraffin embedded tissues section to a stack of membranes which is then probed with antibodies for detection of individual epitopes. This method converts a traditional tissue section into a multiplex platform for expression profiling. A single tissue section can be transferred to up to ten membranes, each of which is probed with different antibodies, and detected with fluorescent secondary antibodies, and quantified by a microarray scanner. Total protein can be determined on each membrane, hence each antibody has its own normalization. This method works with phospho-specific antibodies as well as antibodies that do not readily work well with paraffin embedded tissue. This novel technique enables archival paraffin embedded tissue to be molecularly profiled in a rapid and quantifiable manner, and reduces the tissue microarray to a form of protein array. This method is a new tool for exploration of the vast archive of formalin fixed, paraffin embedded tissue, as well as a tool for translational medicine.

Antibodies, Phospho-Specific↗

PubMed2006

A multiplex tissue immunoblotting assay for proteomic profiling: a pilot study of the normal to tumor transition of esophageal squamous cell carcinoma.

Esophageal cancer remains a highly lethal malignancy for which the genetic and proteomic events are poorly understood. Studies have reported dysregulated proteins in esophageal carcinoma; however, the magnitude of these changes remains largely uncharacterized. Little is known about alterations early in the neoplastic pathway. Using multiplex tissue immunoblotting, we quantified the expression of seven proteins in esophageal carcinogenesis. Regions of normal, dysplasia, and invasive carcinoma of the squamous esophagus in six patients were characterized. Pan-cytokeratin (CK) was essentially unchanged across the transition (0.96 in dysplasia and 0.69 in tumor). Expression levels of annexin 1, CK-4, and CK-14 were all decreased in dysplasia and tumor compared with normal (reference, 1.00): annexin 1, 0.30 in dysplasia and 0.15 in tumor; CK-4, 0.20 in dysplasia and 0.16 in tumor; and CK-14, 0.54 in dysplasia and 0.40 in tumor. Expression of two proteins was increased in dysplasia and tumor versus normal: cyclooxygenase-2, 1.35 in dysplasia and 2.32 in tumor and p53, 1.29 in dysplasia and 2.37 in tumor. Secreted protein, acidic and rich in cysteine, which is expressed in the adjacent stroma, was 1.56-fold higher in stroma underlying dysplasia and 6.20-fold increased in dysplastic stroma surrounding invasive tumor. These findings suggest that changes in protein expression can be detected during the transition to dysplasia and may be useful biomarkers.

Biomarkers, Tumor↗

PubMed2005

Profiling EGFR activity in head and neck squamous cell carcinoma by using a novel layered membrane Western blot technology.

Given the role of epidermal growth factor receptor (EGFR) in head and neck squamous cell carcinomas (HNSCC), several rational approaches have now been utilized to abrogate tyrosine kinase activity and its disengagement from downstream signal transducers. Monitoring the activity of these molecules could potentially be useful to determine not only drug efficacy but also to identify HNSCC patients most likely to benefit from this type of therapy. In this study we have used a novel high throughput multi-layered Western blotting (MLWestern) method that allows the detection of multiple proteins from a single experiment in order to characterize key components in the EGFR signaling pathway in HNSCC cells. Total and activated forms of EGFR and the downstream effectors, Erk and Akt were readily detected in HNSCC cells, where in the control cells (HaCaT) these proteins could only be detected in EGF stimulated cells. Results from conventional Western blot and MLWestern were comparable. Clustering analysis of protein expression revealed similarities in cellular response between some of the cell lines indicative of similarities in their biological response. The data indicate that MLWestern can be potentially applied to identify molecular targets that could be used for rational therapeutic intervention strategies.

Blotting, Western↗

PubMed2005

Layered peptide arrays: high-throughput antibody screening of clinical samples.

High-throughput methods to detect and quantify antibodies in sera and other patient specimens have use for many clinical and laboratory studies, including those associated with cancer detection, microbial exposures, and autoimmune diseases. We developed a new technique, termed layered peptide array (LPA), to serve as a screening tool to detect antibodies in a highly multiplexed format. We demonstrate here that a prototype LPA was capable of producing approximately 5000 measurements per experiment and appeared to be scalable to higher throughput levels. Sera and saliva from Sjögren's syndrome patients served as a test set to examine antibody titers in clinical samples. The LPA platform exhibited both a high sensitivity (100%) and high specificity (94%) for correctly identifying SSB antigen-positive samples. The multiplex capability of the platform was also confirmed when serum and saliva samples were analyzed for antibody reactivity to several peptides, including Sjögren's syndrome antigens A and B. The data indicate that LPA analysis will be a useful method for a number of screening applications.

Antibodies↗

PubMed2005

Pentameric procyanidin from Theobroma cacao selectively inhibits growth of human breast cancer cells.

A naturally occurring, cocoa-derived pentameric procyanidin (pentamer) was previously shown to cause G0/G1 cell cycle arrest in human breast cancer cells by an unknown molecular mechanism. Here, we show that pentamer selectively inhibits the proliferation of human breast cancer cells (MDA MB-231, MDA MB-436, MDA MB-468, SKBR-3, and MCF-7) and benzo(a)pyrene-immortalized 184A1N4 and 184B5 cells. In contrast, normal human mammary epithelial cells in primary culture and spontaneously immortalized MCF-10A cells were significantly resistant. We evaluated whether this differential response to pentamer may involve depolarization of the mitochondrial membrane. Pentamer caused significant depolarization of mitochondrial membrane in MDA MB231 cells but not the more normal MCF-10A cells, whereas other normal and tumor cell lines tested gave variable results. Further investigations, using a proteomics approach with pentamer-treated MDA MB-231, revealed a specific dephosphorylation, without changes in protein expression, of several G1-modulatory proteins: Cdc2 (at Tyr15), forkhead transcription factor (at Ser256, the Akt phosphorylation site) and p53 (Ser392). Dephosphorylation of p53 (at Ser392) by pentamer was confirmed in MDA MB-468 cells. However, both expression and phosphorylation of retinoblastoma protein were decreased after pentamer treatment. Our results show that breast cancer cells are selectively susceptible to the cytotoxic effects of pentameric procyanidin, and suggest that inhibition of cellular proliferation by this compound is associated with the site-specific dephosphorylation or down-regulation of several cell cycle regulatory proteins.

Antioxidants↗

PubMed2005

Novel application of layered expression scanning for proteomic profiling of plucked hair follicles.

BACKGROUND: There is a need for a technology that can quantitatively assay multiple proteins from a single hair follicle while preserving the morphology of the follicle. For proteomic profiling, the technology should be less labor intensive, with a higher throughput, more quantitative and more reproducible than immunohistochemistry. OBJECTIVE: To test the ability of a novel method, layered expression scanning of hair (LES-hair) to detect the levels and localization of proteins in plucked hair follicles. METHODS: LES-hair was used to assay proteins in the plucked hair follicle. RESULTS: LES-hair detected differential expression of proteins within discrete regions of the plucked hair follicle. These proteins included cleaved caspase 3, Ki-67 and the phosphorylated forms of c-Kit, epidermal growth factor receptor and vascular endothelial growth factor receptor. CONCLUSION: LES-hair provides a research tool for studying the basic biology of plucked hair follicles and has potential clinical applications such as monitoring treatment of alopecia or using plucked hair follicles as a surrogate tissue to monitor pharmacodynamic effects of targeted cancer therapies.

Adult↗

PubMed2004

Profiling the expression of mitogen-induced T-cell proteins by using multi-membrane dot-blotting.

High throughput technologies are standard methods for analysis of the proteome. Multi-layer multi-well plate dot-blotting system (MLDot) technology is a high-throughput dot blotting system that provides a simple, cost-effective approach for protein expression profiling in multiple samples. In contrast to traditional dot blot, MLDot uses a layered stack of thin, sieve-like membranes in place of a single nitrocellulose membrane. Therefore, up to 10 membranes can be prepared from the samples arrayed in a single 96-well plate. We describe the ability of MLDot to detect the predicted changes in protein expression following multiple mitogen treatment of T-cells. We compare the levels of the phopshorylated forms of CREB, Jun, and Akt in Jurkat T-cells as detected by MLDot to those measured by a gel-based assay. We also describe the ability of MLDot to detect differences in the levels of phosphorylated Akt in Jurkat cells as compared to primary lymphocytes.

Biotinylation↗

PubMed2004

Modulation of tumor-host interactions, angiogenesis, and tumor growth by tissue inhibitor of metalloproteinase 2 via a novel mechanism.

Solid tumors depend on angiogenesis for sustained growth. Tissue inhibitor of metalloproteinase 2 (TIMP-2) is an angiogenesis inhibitor initially characterized for its ability to block matrix metalloproteinases; however, recent data suggest that the antiangiogenic action of TIMP-2 may rely on matrix metalloproteinase-independent mechanisms. The aim of this study was to identify molecular pathways involved in the effects of TIMP-2 on processes dependent on tumor-host interactions such as angiogenesis. Using in vitro cell culture and a syngeneic murine tumor model, we compared the effects of TIMP-2 overexpression on gene expression profiles in vitro to those observed in vivo. Validating these findings by real-time quantitative PCR and layered protein scanning, we identified up-regulation of mitogen-activated protein kinase phosphatase 1 as an effector of the antiangiogenic function of TIMP-2. Up-regulation of mitogen-activated protein kinase phosphatase 1 in tumors overexpressing TIMP-2 leads to dephosphorylation of p38 mitogen-activated protein kinase and inhibition of tumor growth and angiogenesis. Phosphatase activity appears important in regulating tumor angiogenesis, offering a promising direction for the identification of novel molecular targets and antiangiogenic compounds for the treatment of cancer.

Animals↗

PubMed2004

Direct interaction with Hoxd proteins reverses Gli3-repressor function to promote digit formation downstream of Shh.

Sonic hedgehog (Shh) signaling regulates both digit number and identity, but how different distinct digit types (identities) are specified remains unclear. Shh regulates digit formation largely by preventing cleavage of the Gli3 transcription factor to a repressor form that shuts off expression of Shh target genes. The functionally redundant 5'Hoxd genes regulate digit pattern downstream of Shh and Gli3, through as yet unknown targets. Enforced expression of any of several 5'Hoxd genes causes polydactyly of different distinct digit types with posterior transformations in a Gli3(+) background, whereas, in Gli3 null limbs, polydactylous digits are all similar, short and dysmorphic, even though endogenous 5'Hoxd genes are broadly misexpressed. We show that Hoxd12 interacts genetically and physically with Gli3, and can convert the Gli3 repressor into an activator of Shh target genes. Several 5'Hoxd genes, expressed differentially across the limb bud, interact physically with Gli3. We propose that a varying [Gli3]:[total Hoxd] ratio across the limb bud leads to differential activation of Gli3 target genes and contributes to the regulation of digit pattern. The resulting altered balance between 'effective' Gli3 activating and repressing functions may also serve to extend the Shh activity gradient spatially or temporally.

Animals↗

PubMed2004

A novel technique for quantifying changes in vascular density, endothelial cell proliferation and protein expression in response to modulators of angiogenesis using the chick chorioallantoic membrane (CAM) assay.

Reliable quantitative evaluation of molecular pathways is critical for both drug discovery and treatment monitoring. We have modified the CAM assay to quantitatively measure vascular density, endothelial proliferation, and changes in protein expression in response to anti-angiogenic and pro-angiogenic agents. This improved CAM assay can correlate changes in vascular density with changes seen on a molecular level. We expect that these described modifications will result in a single in vivo assay system, which will improve the ability to investigate molecular mechanisms underlying the angiogenic response.

Journal Article↗

PubMed2004

Ventral tail bud mesenchyme is a signaling center for tail paraxial mesoderm induction.

A large body of evidence from several systems indicates that formation of the vertebrate tail is morphogenetically continuous with gastrulation, including neural inducing activity in descendants of the gastrula organizer. However, the signaling centers and molecular events regulating tail mesoderm induction and its organized elongation remain poorly defined. In mammals, the ventral ectoderm ridge (VER) is essential to maintain ongoing formation of paraxial mesoderm and somitogenesis in cultures of intact tail. Avian tail buds contain a similar VER structure. Here, we report that the chick ventral tail bud operates as a signaling center for paraxial mesoderm induction. By using "organizer" style grafting assays to early host embryos, we found that ventral tail bud was able to induce elongated paraxial mesodermal extensions and that the ventral tail bud mesenchyme underlying the VER is both necessary and sufficient for the induction in this assay system. Our observations combined with those of others suggest that interplay between several different signaling centers in the amniote tail bud regulates the coordinate induction and elongation of axial and paraxial structures in the developing tail.

Animals↗

PubMed2004

Multimembrane dot-blotting: a cost-effective tool for proteome analysis.

The molecular profiles of protein expression from hundreds of cell lysates can be determined in a high-throughput manner by using fluorescent bead technologies, enzyme-linked immunosorbent assays (ELISAs), and protein microarrays. Although powerful, these tools are costly and technically challenging and thus have limited accessibility for many research groups. We propose a modification of traditional dot blotting that increases throughput of this approach and provides a simple and cost-effective technique for profiling multiple samples. In contrast to traditional blotting that uses a single membrane, we introduce blotting onto a stack of novel, thin, sieve-like membranes. These membranes have a high affinity for binding proteins, but have a lower capacity of protein binding compared to traditional (nitrocellulose) membranes. We compare the linear binding capacity and variability of these novel membranes with nitrocellulose membranes. Also, we describe the use of these membranes in a multilayer dot blot format for profiling mitogen-mediated signal transduction pathways in T cells.

Blood Proteins↗

PubMed2003

A role for the mesenchymal T-box gene Brachyury in AER formation during limb development.

During limb development, several signaling centers organize limb pattern. One of these, the apical ectodermal ridge (AER), is critical for proximodistal limb outgrowth mediated by FGFs. Signals from the underlying mesoderm, including WNTs and FGFs, regulate early steps of AER induction. Ectodermal factors, particularly En1, play a critical role in regulating morphogenesis of a mature, compact AER along the distal limb apex, from a broad ventral ectodermal precursor domain. Contribution of mesodermal factors to the morphogenesis of a mature AER is less clear. We previously noted that the chick T gene (Brachyury), the prototypical T-box transcription factor, is expressed in the limb bud as well as axial mesoderm and primitive streak. Here we show that T is expressed in lateral plate mesoderm at the onset of limb bud formation and subsequently in the subridge mesoderm beneath the AER. Retroviral misexpression of T in chick results in anterior extension of the AER and subsequent limb phenotypes consistent with augmented AER extent and function. Analysis of markers for functional AER in mouse T(-/-) null mutant limb buds reveals disrupted AER morphogenesis. Our data also suggest that FGF and WNT signals may operate both upstream and downstream of T. Taken together, the results show that T plays a role in the regulation of AER formation, particularly maturation, and suggest that T may also be a component of the epithelialmesenchymal regulatory loop involved in maintenance of a mature functioning AER.

Animals↗

PubMed2003

Layered expression scanning: multiplex analysis of RNA and protein gels.

Northern blots and immunoblots are utilized in laboratories worldwide and offer several important features for analyzing mRNA and protein expression, including accuracy, low cost, evaluation of probe specificity, and information on transcript and protein forms based on molecular size. However, standard blotting techniques are hampered by three factors. They require a significant amount of input material, are laborious, and are capable of measuring only one protein or transcript at a time. Here we describe a simple yet effective technique for the multiplex analysis of standard RNA and protein gels using the layered expression scanning platform. The method relies on a novel membrane with high-affinity low-capacity binding characteristics. Using this approach, multiple blots from an RNA or protein electrophoresis gel can be simultaneously produced. We believe this method will be widely applicable to expression studies for a broad range of biological systems.

Blotting, Northern↗

PubMed2002

Post-analysis follow-up and validation of microarray experiments.

Measurement of gene-expression profiles using microarray technology is becoming increasingly popular among the biomedical research community. Although there has been great progress in this field, investigators are still confronted with a difficult question after completing their experiments: how to validate the large data sets that are generated? This review summarizes current approaches to verifying global expression results, discusses the caveats that must be considered, and describes some methods that are being developed to address outstanding problems.

Animals↗

PubMed2002

Construction of cDNA libraries from microdissected benign and malignant thyroid tissue.

cDNA libraries were constructed from thyroid epithelial cells gained by laser capture microdissection for gene expression analysis of the progression of thyroid cancer. Six histologically diverse thyroid tissue specimens were used. A mean of 93 ng of total RNA was gained per tissue sample from a mean estimated number of 25,000 microdissected cells per sample. Analysis of randomly selected clones from six libraries showed an average insert size of 600 (range, 300-1500) bp. Preliminary sequencing of clones selected from the six libraries indicates a range of 46% to 62% known genes per library, 4% to 25% anonymous expressed sequence tags per library, and 15% to 43% novel expressed sequence tags per library. Thyroglobulin was found in normal thyroid epithelium and follicular thyroid adenoma, whereas calcitonin precursor transcripts were found in medullary thyroid carcinoma. We demonstrate production of high-quality cDNA libraries of microdissected tissue of the thyroid, which should prove useful for gene expression analysis of human thyroid tumors.

Adenoma↗