Biomedical subjects

Samir M Hanash

Publications and source records attributed to Samir M Hanash.

At least 19 recordsLinked to original sources

PubMed2006

Modification of host lipid raft proteome upon hepatitis C virus replication.

Hepatitis C virus (HCV) replication complex resides in detergent-insoluble subcellular domains or lipid rafts. We used two proteomics approaches to characterize the protein content of lipid rafts isolated from Huh7 cells and its modification upon HCV replication. Using two-dimensional electrophoresis and mass spectrometry, we identified approximately 100 protein spots in the isolated lipid rafts; among them, 39 were reproducibly modified in HCV replicon cell lines as compared with control cell lines. We also used stable isotope labeling by amino acids in cell culture (SILAC) combined with one-dimensional electrophoresis separation and mass spectrometry. Using this approach, we identified 1036 individual proteins based on peptides selected with at least 95% confidence; among them, 413 proteins were identified with at least two peptides. Quantification analysis identified 150 proteins modified by at least 2.5-fold (110 up-regulated and 40 down-regulated) in HCV-replicating cells compared with controls. Protein identifications and quantifications obtained by both proteomics approaches were largely concordant. Modulated proteins included a majority of proteins involved in vesicular and protein trafficking and in cell signaling. Remarkably for a large number of proteins, their up-regulation in lipid rafts of HCV replicon cells was due to their relocalization. By using small interfering RNAs directed to the modulated small GTPases Cdc42 and RhoA, we observed an increase in HCV replication, whereas reduction of syntaxin 7 expression resulted in decreased replication of HCV. Our findings indicate that protein subcellular relocalization occurs in HCV-containing cells that can directly affect HCV replication.

Cell Extracts↗

PubMed2006

Cellular retinoic acid-binding protein II is a direct transcriptional target of MycN in neuroblastoma.

Neuroblastoma is a heterogeneous disease in which 22% of tumors show MycN oncogene amplification and are associated with poor clinical outcome. MycN is a transcription factor that regulates the expression of a number of proteins that affect the clinical behavior of neuroblastoma. We report here that cellular retinoic acid-binding protein II (CRABP-II) is a novel MycN target, expressed at significantly higher levels in primary neuroblastoma tumors with mycN oncogene amplification as compared with non-MycN-amplified tumors. Moreover, regulated induction and repression of MycN in a neuroblastoma-derived cell line resulted in temporal and proportionate expression of CRABP-II. CRABP-II is expressed in several cancers, but its role in tumorigenesis has not been elucidated. We show that MycN binds to the promoter of CRABP-II and induces CRABP-II transcription directly. In addition, CRABP-II-transfected neuroblastoma cell lines show an increase in MycN protein levels resulting in increased cell motility. Gene expression profiling of CRABP-II-expressing cell lines uncovered increased expression of the HuB (Hel N1) gene. Hu proteins have been implicated in regulating the stability of MycN mRNA and other mRNAs by binding to their 3' untranslated regions. We did not, however, observe any change in MycN mRNA stability or protein half-life in response to CRABP-II expression. In contrast, de novo MycN protein synthesis was increased in CRABP-II-expressing neuroblastoma cells, thereby suggesting an autoregulatory loop that might exacerbate the effects of MycN gene amplification and affect the clinical outcome. Our findings also suggest that CRABP-II may be a potential therapeutic target for neuroblastoma.

Binding Sites↗

PubMed2006

Challenges in deriving high-confidence protein identifications from data gathered by a HUPO plasma proteome collaborative study.

The Human Proteome Organization (HUPO) recently completed the first large-scale collaborative study to characterize the human serum and plasma proteomes. The study was carried out in different locations and used diverse methods and instruments to compare and integrate tandem mass spectrometry (MS/MS) data on aliquots of pooled serum and plasma from healthy subjects. Liquid chromatography (LC)-MS/MS data sets from 18 laboratories were matched to the International Protein Index database, and an initial integration exercise resulted in 9,504 proteins identified with one or more peptides, and 3,020 proteins identified with two or more peptides. This article uses a rigorous statistical approach to take into account the length of coding regions in genes, and multiple hypothesis-testing techniques. On this basis, we now present a reduced set of 889 proteins identified with a confidence level of at least 95%. We also discuss the importance of such an integrated analysis in providing an accurate representation of a proteome as well as the value such data sets contain for the high-confidence identification of protein matches to novel exons, some of which may be localized in alternatively spliced forms of known plasma proteins and some in previously nonannotated gene sequences.

Blood Proteins↗

PubMed2006

Long-term ethanol consumption alters pancreatic gene expression in rats: a possible connection to pancreatic injury.

OBJECTIVES: Long-term ethanol consumption does not cause acute pancreatitis but rather sensitizes the pancreas to subsequent insults. The mechanisms responsible for this sensitization are unknown. To determine whether alterations in pancreatic gene expression might participate in ethanol-mediated sensitization, we performed gene-profiling analysis. METHODS: Animals were fed ethanol-containing Lieber-DeCarli or control diet (pair-fed). After 8 weeks, pancreatic RNA expression was analyzed using Affimetrix GeneChips. Changes in specific genes were verified using quantitative reverse transcriptase-polymerase chain reaction. RESULTS: Long-term ethanol feeding caused a significant alteration of pancreatic gene expression. Selection criteria of changes more than 3-fold and P < 0.05 yielded 114 probe sets. Activating transcription factor 3, heat shock protein 70, heat shock protein 27, and mesotrypsinogen were increased, whereas pancreatitis associate protein, folate carrier, and metallothionein were decreased. CONCLUSIONS: Ethanol had a profound effect on pancreatic gene expression. The genes identified as elevated and reduced in this study may contribute to pancreatic sensitivity to stress. This study indicates for the first time the identities of multiple genes whose expression levels are dramatically influenced by long-term ethanol feeding. The identified genes may help explain the relationship between long-term ethanol abuse and pancreatic disease and lead to possible preventative or therapeutic approaches to ethanol-induced pancreatic disease.

Activating Transcription Factor 3↗

PubMed2005

Distinctive serum protein profiles involving abundant proteins in lung cancer patients based upon antibody microarray analysis.

BACKGROUND: Cancer serum protein profiling by mass spectrometry has uncovered mass profiles that are potentially diagnostic for several common types of cancer. However, direct mass spectrometric profiling has a limited dynamic range and difficulties in providing the identification of the distinctive proteins. We hypothesized that distinctive profiles may result from the differential expression of relatively abundant serum proteins associated with the host response. METHODS: Eighty-four antibodies, targeting a wide range of serum proteins, were spotted onto nitrocellulose-coated microscope slides. The abundances of the corresponding proteins were measured in 80 serum samples, from 24 newly diagnosed subjects with lung cancer, 24 healthy controls, and 32 subjects with chronic obstructive pulmonary disease (COPD). Two-color rolling-circle amplification was used to measure protein abundance. RESULTS: Seven of the 84 antibodies gave a significant difference (p < 0.01) for the lung cancer patients as compared to healthy controls, as well as compared to COPD patients. Proteins that exhibited higher abundances in the lung cancer samples relative to the control samples included C-reactive protein (CRP; a 13.3 fold increase), serum amyloid A (SAA; a 2.0 fold increase), mucin 1 and alpha-1-antitrypsin (1.4 fold increases). The increased expression levels of CRP and SAA were validated by Western blot analysis. Leave-one-out cross-validation was used to construct Diagonal Linear Discriminant Analysis (DLDA) classifiers. At a cutoff where all 56 of the non-tumor samples were correctly classified, 15/24 lung tumor patient sera were correctly classified. CONCLUSION: Our results suggest that a distinctive serum protein profile involving abundant proteins may be observed in lung cancer patients relative to healthy subjects or patients with chronic disease and may have utility as part of strategies for detecting lung cancer.

Antibodies↗

PubMed2005

Intact-protein-based high-resolution three-dimensional quantitative analysis system for proteome profiling of biological fluids.

The substantial complexity and vast dynamic range of protein abundance in biological fluids, notably serum and plasma, present a formidable challenge for comprehensive protein analysis. Integration of multiple technologies is required to achieve high-resolution and high-sensitivity proteomics analysis of biological fluids. We have implemented an orthogonal three-dimensional intact-protein analysis system (IPAS), coupled with protein tagging and immunodepletion of abundant proteins, to quantitatively profile the human plasma proteome. Following immunodepletion, plasma proteins in each of paired samples are concentrated and labeled with a different Cy dye, before mixing. Proteins are subsequently separated in three dimensions according to their charge, hydrophobicity, and molecular mass. Differences in the abundance of resolved proteins are determined based on Cy dye ratios. We have applied this strategy to profile the plasma proteome for changes that occur with acute graft-versus-host disease (GVHD), following allogeneic bone marrow transplantation (BMT). Using capillary HPLC ESI Q-TOF MS, we identified 75 proteins in the micromolar to femtomolar range that exhibited quantitative differences between the pre- and post-GVHD samples. These proteins included serum amyloid A, apolipoproteins A-I/A-IV, and complement C3 that are well-known acute-phase reactants likely reflecting the post-BMT inflammatory state. In addition, we identified some potentially interesting immunologically relevant molecules including vitamin D-binding protein, fetuin, vitronectin, proline-rich protein 3 and 4, integrin-alpha, and leukocyte antigen CD97. IPAS provides a combination of comprehensive profiling and quantitative analysis, with a substantial dynamic range, for disease-related applications.

Adult↗

PubMed2005

Methods of comparative proteomic profiling for disease diagnostics.

The recent development of numerous technologies for proteome analysis holds the promise of new and more precise methods for disease diagnosis. In this review, we provide an overview of some of these technologies including two-dimensional gel electrophoresis (2DE), historically the workhorse of proteomic analysis, as well as some newer approaches such as liquid phase separations combined with mass spectrometry, and protein microarrays. It is evident that each method has its own strengths and weaknesses and no single method will be optimal in all applications. However, the continuing development of innovative strategies for protein separation and analysis is providing a wealth of new tools for multi-dimensional protein profiling that will advance our capabilities in disease diagnostics and our understanding of disease pathology.

Chromatography, Liquid↗

PubMed2005

A wide range of protein isoforms in serum and plasma uncovered by a quantitative intact protein analysis system.

We have implemented an orthogonal 3-D intact protein analysis system (IPAS) to quantitatively profile protein differences between human serum and plasma. Reference specimens consisting of pooled Caucasian-American serum, citrate-anticoagulated plasma, and EDTA-anticoagulated plasma were each depleted of six highly abundant proteins, concentrated, and labeled with a different Cy dye (Cy5, Cy3, or Cy2). A mixture consisting of each of the labeled samples was subjected to three dimensions of separation based on charge, hydrophobicity, and molecular mass. Differences in the abundance of proteins between each of the three samples were determined. More than 5000 bands were found to have greater than two-fold difference in intensity between any pair of labeled specimens by quantitative imaging. As expected, some of the differences in band intensities between serum and plasma were attributable to proteins related to coagulation. Interestingly, many proteins were identified in multiple fractions, each exhibiting different pI, hydrophobicity, or molecular mass. This is likely reflective of the expression of different protein isoforms or specific protein cleavage products, as illustrated by complement component 3 precursor and clusterin. IPAS provides a high resolution, high sensitivity, and quantitative approach for the analysis of serum and plasma proteins, and allows assessment of PTMs as a potential source of biomarkers.

Anticoagulants↗

PubMed2005

A functional annotation of subproteomes in human plasma.

The data collected by Human Proteome Organization's Plasma Proteome Pilot project phase was analyzed by members of our working group. Accordingly, a functional annotation of the human plasma proteome was carried out. Here, we report the findings of our analyses. First, bioinformatic analyses were undertaken to determine the likely sources of plasma proteins and to develop a protein interaction network of proteins identified in this project. Second, annotation of these proteins was performed in the context of functional subproteomes involved in the coagulation pathway, the mononuclear phagocytic system, the inflammation pathway, the cardiovascular system, and the liver; as well as the subset of proteins associated with DNA binding activities. Our analyses contributed to the Plasma Proteome Database (http://www.plasmaproteomedatabase.org), an annotated database of plasma proteins identified by HPPP as well as from other published studies. In addition, we address several methodological considerations including the selective enrichment of post-translationally modified proteins by the use of multi-lectin chromatography as well as the use of peptidomic techniques to characterize the low molecular weight proteins in plasma. Furthermore, we have performed additional analyses of peptide identification data to annotate cleavage of signal peptides, sites of intra-membrane proteolysis and post-translational modifications. The HPPP-organized, multi-laboratory effort, as described herein, resulted in much synergy and was essential to the success of this project.

Blood Coagulation↗

PubMed2005

Overview of the HUPO Plasma Proteome Project: results from the pilot phase with 35 collaborating laboratories and multiple analytical groups, generating a core dataset of 3020 proteins and a publicly-available database.

HUPO initiated the Plasma Proteome Project (PPP) in 2002. Its pilot phase has (1) evaluated advantages and limitations of many depletion, fractionation, and MS technology platforms; (2) compared PPP reference specimens of human serum and EDTA, heparin, and citrate-anti-coagulated plasma; and (3) created a publicly-available knowledge base (www.bioinformatics.med.umich.edu/hupo/ppp; www.ebi.ac.uk/pride). Thirty-five participating laboratories in 13 countries submitted datasets. Working groups addressed (a) specimen stability and protein concentrations; (b) protein identifications from 18 MS/MS datasets; (c) independent analyses from raw MS-MS spectra; (d) search engine performance, subproteome analyses, and biological insights; (e) antibody arrays; and (f) direct MS/SELDI analyses. MS-MS datasets had 15 710 different International Protein Index (IPI) protein IDs; our integration algorithm applied to multiple matches of peptide sequences yielded 9504 IPI proteins identified with one or more peptides and 3020 proteins identified with two or more peptides (the Core Dataset). These proteins have been characterized with Gene Ontology, InterPro, Novartis Atlas, OMIM, and immunoassay-based concentration determinations. The database permits examination of many other subsets, such as 1274 proteins identified with three or more peptides. Reverse protein to DNA matching identified proteins for 118 previously unidentified ORFs. We recommend use of plasma instead of serum, with EDTA (or citrate) for anticoagulation. To improve resolution, sensitivity and reproducibility of peptide identifications and protein matches, we recommend combinations of depletion, fractionation, and MS/MS technologies, with explicit criteria for evaluation of spectra, use of search algorithms, and integration of homologous protein matches. This Special Issue of PROTEOMICS presents papers integral to the collaborative analysis plus many reports of supplementary work on various aspects of the PPP workplan. These PPP results on complexity, dynamic range, incomplete sampling, false-positive matches, and integration of diverse datasets for plasma and serum proteins lay a foundation for development and validation of circulating protein biomarkers in health and disease.

Algorithms↗

PubMed2005

Analysis of tumor-host interactions by gene expression profiling of lung adenocarcinoma xenografts identifies genes involved in tumor formation.

Tumor cell lines are relied on extensively for cancer investigations, yet cultured cells in an in vitro environment differ considerably in behavior compared with those of the same cancer cells that proliferate and form tumors in vivo. To uncover gene expression changes related to tumor formation, gene expression profiles of human lung adenocarcinoma (A549) cells grown as lung tumors in immune-compromised mice were compared with profiles of the same cells grown in vitro. Additionally, profiles of uninvolved adjacent mouse tissue were determined. A profound interplay between cancer cells and the host was shown that affected a complex protein interaction network involving processes of extracellular interaction, growth factor signaling, hemostasis, immune response, and transcriptional regulation. Growth in vivo of A549 cells, which carry an activating k-ras mutation, induced changes in gene expression that corresponded highly to a pattern characteristic of human lung tumors with k-ras mutation. Cytokines interleukin-4, interleukin-6, and IFN-gamma each induced distinct in vitro genomic responses in cancer cells that emulated many of the changes in gene expression observed in vivo. Genes that were both selectively induced in vivo and overexpressed in human lung adenocarcinoma tumors included CSPG2, which has not been associated previously with tumor formation. Knockdown in A549 of CSPG2 by RNA interference significantly inhibited tumor growth in vivo but not in vitro. Thus, analysis of tumor xenografts by gene expression profiling has the potential for identifying genes involved in tumor development that may not be expressed in cancer cells grown in vitro.

Adenocarcinoma↗

PubMed2005

Oligonucleotide-directed microarray gene profiling of pancreatic adenocarcinoma.

Successful gene profiling studies involve careful experimental design, use of sensitive and accurate technologies, and statistically valid analysis of experimental results. In this chapter we describe our approach to the profiling of pancreatic adenocarcinoma to illustrate the various steps and methods involved in this type of study. Pancreatic adenocarcinoma is a particularly challenging subject for gene profiling, as these tumors have a profound desmoplastic response such that neoplastic epithelium makes up only a small proportion of the tissue mass. We have utilized statistical comparisons of gene expression between adenocarcinoma, normal pancreas, samples of chronic pancreatitis, and pancreatic cancer cell lines that provides a means to deduct the influence of the stromal elements. We utilized oligonucleotide-directed gene chips (Affymetrix), as they allow the simultaneous interrogation of thousands of genes in an efficient, reproducible, sensitive, and highly quantitative manner. The details of the approach we utilized are reported here, including information on experimental design, sample collection, expression level measurements, and data analysis for gene profiling.

Adenocarcinoma↗

PubMed2004

Identification of defensin alpha6 as a potential biomarker in colon adenocarcinoma.

There is substantial interest in the identification of circulating human tumor-derived proteins in serum for the purposes of early cancer diagnosis. We have implemented an approach based on the analysis of microarray data for the identification of tumor proteins that may have utility as biomarkers in colon cancer. Expression analysis of microarray data obtained from a variety of 283 tumors and normal tissues revealed that defensin alpha6 was maximally expressed in colon cancer. These findings were corroborated by reverse transcription-PCR, in which the colon cancer cell lines LoVo, Caco2, HCT-15, SW480, and SW620 showed significantly higher levels of defensin alpha6 expression than did non-colon cancer cell lines. Moreover, our data were concordant with data obtained from the NCI, National Institutes of Health Cancer Genome Anatomy Project. To evaluate defensin alpha6 as a potential biomarker of colon cancer, a preliminary "training" set of serum from 91 healthy donors and 109 colon cancer patients was analyzed by enzyme-linked immunosorbent assay. The data pattern was confirmed by an independent set of 67 masked serum samples: 18 from healthy donors and 49 from colon cancer patients. This result yielded a sensitivity of 69.4% (95% CI 54.6-81.8), specificity of 83.3% (58.6-96.4), and positive predictive value of 91.9% (78.1-98.3). These findings justify a prospective assessment of serum defensin alpha6 protein as a screening tool for colon cancer.

Adenocarcinoma↗

PubMed2004

An autoantibody-mediated immune response to calreticulin isoforms in pancreatic cancer.

The identification of circulating tumor antigens or their related autoantibodies provides a means for early cancer diagnosis as well as leads for therapy. We have used a proteomic approach to identify proteins that commonly induce a humoral response in pancreatic cancer. Aliquots of solubilized proteins from a pancreatic cancer cell line (Panc-1) were subjected to two-dimensional PAGE, followed by Western blot analysis in which sera of individual patients were tested for primary antibodies. Sera from 36 newly diagnosed patients with pancreatic cancer, 18 patients with chronic pancreatitis, 33 patients with other cancers, and 15 healthy subjects were analyzed. Autoantibodies were detected against either one or two calreticulin isoforms identified by mass spectrometry in sera from 21 of 36 patients with pancreatic cancer. One of 18 chronic pancreatitis patients and 1 of 15 healthy controls demonstrated autoantibodies to calreticulin isoform 1; none demonstrated autoantibodies to isoform 2. None of the sera from patients with colon cancer exhibited reactivity against either of these two proteins. One of 14 sera from lung adenocarcinoma patients demonstrated autoantibodies to calreticulin isoform 1; 2 of 14 demonstrated autoantibodies to isoform 2. Immunohistochemical analysis of calreticulin in pancreatic/ampullary tumor tissue arrays using an isoform nonspecific antibody revealed diffuse and consistent cytoplasmic staining in the neoplastic epithelial cells of the pancreatic and ampullary adenocarcinomas. The detection of autoantibodies to calreticulin isoforms may have utility for the early diagnosis of pancreatic cancer.

Adenocarcinoma↗

PubMed2004

The bioenergetic signature of lung adenocarcinomas is a molecular marker of cancer diagnosis and prognosis.

The aim of this study was to investigate the mitochondrial bioenergetic signature of lung adenocarcinomas as a prognostic marker of cancer progression. For this purpose, a series of 90 lung adenocarcinomas and 10 uninvolved lung samples were examined for quantitative differences in protein expression using two-dimensional polyacrylamide gel electrophoresis. The beta subunit of the mitochondrial H(+)-ATP synthase (beta-F1-ATPase) and heat shock protein 60 (Hsp 60), and the glycolytic glyceraldehyde-3-phosphate dehydrogenase (GAPDH), used to define the bioenergetic cellular (BEC) index, were identified using mass spectrometry and specific antibodies. Correlations of the expression level of the protein markers and of the BEC index were established with the clinicopathological information of the tumors and the follow-up data of the patients. The expression of beta-F1-ATPase is significantly reduced in lung adenocarcinomas in the absence of significant changes in the expression of Hsp 60 and of a major GAPDH isoform. Cross-validation analysis using the beta-F1-ATPase/Hsp 60 ratio and GAPDH expression as predictor variables revealed a classification sensitivity of 97.3%. The beta-F1-ATPase/Hsp 60 ratio is significantly higher in well differentiated and bronchioloalveolar tumors than in moderate or poorly differentiated and in bronchial-derived tumors. The BEC index of T1 tumors was significantly higher than that of T2 tumors. Likewise, stage IA tumors had a higher BEC index than stage IB tumors. Kaplan-Meier survival analysis using the BEC index as predictor of survival revealed that within tumors of the same size or stage I or with no lymph node metastasis (N0) the patients bearing 'low' BEC index tumors had a significant worse prognosis. We conclude that the bioenergetic signature of lung adenocarcinomas is altered, further providing a relevant marker for the diagnosis and classification of lung adenocarcinomas, and for the prognosis of lung cancer patients.

Adenocarcinoma↗

PubMed2004

Reduced selenium-binding protein 1 expression is associated with poor outcome in lung adenocarcinomas.

The effects of selenium, an essential nutrient with anti-carcinogenic properties, are mediated by selenium-binding proteins. The protein expression status of human selenium-binding protein 1 (SBP1) in human tumours and the exact function of this protein are not known. In this study, quantitative two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) was used on 93 lung adenocarcinomas and ten uninvolved lung samples. Two likely isoforms of a 56 kD protein that showed a significantly decreased abundance in lung adenocarcinomas were observed. Tandem mass spectrometry and 2-D western blot analysis identified these two proteins as human SBP1. Tumour tissue microarrays were utilized to examine the cellular expression patterns of SBP1 using immunohistochemistry. The same tissue samples were examined for SBP1 mRNA expression using oligonucleotide microarrays. Two major SBP1 isoforms were detected, with an acidic isoform (457) being significantly down-regulated in lung adenocarcinomas compared with normal lung (p = 0.02). Two additional more acidic SBP1 isoforms were only observed in normal lung. SBP1 protein isoforms and SBP1 mRNA levels were significantly decreased in poorly differentiated (versus moderately and well-differentiated), T2-T4 (versus T1), and bronchus-derived (versus bronchioloalveolar) tumours. Low levels of SBP1 protein (native form, 460) correlated significantly with poor survival (p = 0.007). The lack of SBP1 expression was not due to gene deletion. Treatment of A549 lung adenocarcinoma cells with the methylation inhibitor 5-azacytidine did not affect expression of the SBP1 protein. Analysis of the tumour proliferation status using Ki-67 suggests that down-regulated expression of SBP1 may reflect increased cell proliferation and decreased differentiation in lung adenocarcinomas.

Adenocarcinoma↗

PubMed2004

Application of proteomic technologies to tumor analysis.

The sequencing of the human genome has had an enormous impact on the proteomic analysis of cancer by providing a sequence-based framework for understanding the human proteome of tumor cells, tissues, and biological fluids. There is intense interest in applying proteomic technologies to uncover, at the protein level, processes involved in neoplastic transformation and new biomarkers that correlate with early diagnosis, as well as to accelerate the development of new therapeutic targets. To that effect, new technologies are being developed in order to meet the needs for the high throughput and high sensitivity that is required for cancer-related applications of proteomics. These innovative technologies have greatly enhanced our ability to separate and characterize complex protein mixtures, and have aided our ability to identify proteins with greater sensitivity, thereby providing the groundwork for future scientific breakthroughs and possibly providing impetus for the development of personalized cancer therapy.

Animals↗

PubMed2004

Genomic and proteomic analyses of vascular endothelial growth factor and insulin-like growth factor-binding protein 3 in lung adenocarcinomas.

Vascular endothelial growth factor (VEGF) is regulated by the hypoxia-inducible factor 1 (HIF1) pathway and is implicated in tumor progression and patient survival in many types of cancer. Insulin-like growth factor-binding protein 3 (IGFBP3) is also regulated by HIF1 but works in a completely different manner by modulating the activities of insulin-like growth factors and inducing apoptosis. In this study, 2-dimensional (2D) polyacrylamide gel electrophoresis (PAGE) was used to analyze the protein expression profiles of VEGF and IGFBP3 isoforms in 93 lung adenocarcinomas and 10 uninvolved lung samples. The same samples were examined for messenger RNA (mRNA) expression with use of oligonucleotide arrays. Correlation analysis in the lung adenocarcinomas between mRNA expression levels of VEGF and all 4966 other genes was used to identify other biologic processes that may be associated with increased VEGF expression. Two-dimensional gel separations revealed 7 VEGF protein isoforms and 5 isoforms of IGFBP3. VEGF and IGFBP3 mRNA were found to be overexpressed in bronchial-derived lung adenocarcinomas (P < 0.0001), and expression was decreased in well-differentiated lung adenocarcinomas (P < 0.0002). There was a significant correlation (P < 0.01) between VEGF and IGFBP3 mRNA in lung adenocarcinomas; however, no correlation was detected in uninvolved lung samples. Forty genes were identified as the most significantly associated with VEGF expression (r > 0.38, P < 0.001), 17 of which were also associated with IGFBP3, and 12 were known to be induced through the HIF1 pathway. Among other highly correlated genes, several, including bradykinin receptor B2, suggest additional cellular processes that were not previously known to be associated with VEGF expression in lung adenocarcinoma.

Adenocarcinoma↗