Signal pathway profiling of ovarian cancer from human tissue specimens using reverse-phase protein microarrays

Defects in cell signaling pathways play a central role in cancer cell growth, survival, invasion and metastasis. An important goal of proteomics is to characterize and develop "circuit maps" of these signaling pathways in normal and diseased cells. We have used reverse-phase protein array technology coupled with laser capture microdissection and phospho-specific antibodies to examine the activation status of several key molecular "gates" involved in cell survival and proliferation signaling in human ovarian tumor tissue. The levels of activated extracellular-regulated kinase (ERK1/2) varied considerably in tumors of the same histotype, but no significant differences between histotypes were observed. Advanced stage tumors had slightly higher levels of phosphorylated ERK1/2 compared to early stage tumors. The activation status of Akt and glycogen synthase kinase 3beta, key proteins and indicators of the state of the phosphatidylinositol 3-kinase/Akt pro-survival pathway also showed more variation within each histotype than between the histotypes studied. Our results demonstrate the utility of reverse phase protein microarrays for the multiplexed analysis of signal transduction from discreet cell populations of cells procured directly from human ovarian tumor specimens and suggest that patterns in signal pathway activation in ovarian tumors may be patient-specific rather than type or stage specific.     

CGH microarrays and cancer

Genetic alterations are a key feature of cancer cells and typically target biological processes and pathways that contribute to cancer pathogenesis. Array-based comparative genomic hybridization (aCGH) has provided a wealth of new information on copy number changes in cancer on a genome-wide level and aCGH data have also been utilized in cancer classification. More importantly, aCGH analyses have allowed highly accurate localization of specific genetic alterations that, for example, are associated with tumor progression, therapy response, or patient outcome. The genes involved in these aberrations are likely to contribute to cancer pathogenesis, and the high-resolution mapping by aCGH greatly facilitates the subsequent identification of these cancer-associated genes.     

Identification of new autoantigens for primary biliary cirrhosis using human proteome microarrays

Primary biliary cirrhosis (PBC) is a chronic cholestatic liver disease of unknown etiology and is considered to be an autoimmune disease. Autoantibodies are important tools for accurate diagnosis of PBC. Here, we employed serum profiling analysis using a human proteome microarray composed of about 17,000 full-length unique proteins and identified 23 proteins that correlated with PBC. To validate these results, we fabricated a PBC-focused microarray with 21 of these newly identified candidates and nine additional known PBC antigens. By screening the PBC microarrays with additional cohorts of 191 PBC patients and 321 controls (43 autoimmune hepatitis, 55 hepatitis B virus, 31 hepatitis C virus, 48 rheumatoid arthritis, 45 systematic lupus erythematosus, 49 systemic sclerosis, and 50 healthy), six proteins were confirmed as novel PBC autoantigens with high sensitivities and specificities, including hexokinase-1 (isoforms I and II), Kelch-like protein 7, Kelch-like protein 12, zinc finger and BTB domain-containing protein 2, and eukaryotic translation initiation factor 2C, subunit 1. To facilitate clinical diagnosis, we developed ELISA for Kelch-like protein 12 and zinc finger and BTB domain-containing protein 2 and tested large cohorts (297 PBC and 637 control sera) to confirm the sensitivities and specificities observed in the microarray-based assays. In conclusion, our research showed that a strategy using high content protein microarray combined with a smaller but more focused protein microarray can effectively identify and validate novel PBC-specific autoantigens and has the capacity to be translated to clinical diagnosis by means of an ELISA-based method.     

High-throughput proteomics using antibody microarrays

Antibody-based microarrays are a novel technology that hold great promise in proteomics. Microarrays can be printed with thousands of recombinant antibodies carrying the desired specificities, the biologic sample (e.g., an entire proteome) and any specifically bound analytes detected. The microarray patterns that are generated can then be converted into proteomic maps, or molecular fingerprints, revealing the composition of the proteome. Using this tool, global proteome analysis and protein expression profiling will thus provide new opportunities for biomarker discovery, drug target identification and disease diagnostics, as well as providing insights into disease biology. Intense work is currently underway to develop this novel technology platform into the high-throughput proteomic tool required by the research community.     

High-throughput proteomics using antibody microarrays

Antibody-based microarrays are a novel technology that hold great promise in proteomics. Microarrays can be printed with thousands of recombinant antibodies carrying the desired specificities, the biologic sample (e.g., an entire proteome) and any specifically bound analytes detected. The microarray patterns that are generated can then be converted into proteomic maps, or molecular fingerprints, revealing the composition of the proteome. Using this tool, global proteome analysis and protein expression profiling will thus provide new opportunities for biomarker discovery, drug target identification and disease diagnostics, as well as providing insights into disease biology. Intense work is currently underway to develop this novel technology platform into the high-throughput proteomic tool required by the research community.     

Identification of changes in gene loci potentially associated with cervical cancer using NotI microarrays

The investigation of the cancer-associated structural and epigenetic changes in cell genome is a major approach for understanding mechanisms of cancerogenesis. To investigate these genome changes, novel technique of microarrays comprising NotI-linking genome clones was developed. Twenty eight samples from patients with cervical cancer were analyzed using NotI microarrays of human chromosome 3. Deletions, amplifications and methylation were detected for 109 out of 182 NotI clones with different frequency. Notably, 17 NotI-linking clones showed genomic changes in more than 35% of tumor samples investigated, which suggests involvement of genes associated with these clones in development of cervical cancer.     

DNA microarrays in clinical cancer research

The recent sequencing of the human genome, coupled with advances in biotechnology, is enabling the comprehensive molecular "profiling" of human tissues. In particular, DNA microarrays are powerful tools for obtaining global views of human tumor gene expression. Complex information from tumor "expression profiling" studies can, in turn, be used to create novel molecular cancer diagnostics. We discuss the utility of DNA microarray-based tumor profiling in clinical cancer research, highlight some important recent studies, and identify future avenues of research in this evolving field.     

Expression analysis of S100 proteins and RAGE in human tumors using tissue microarrays

Microarray technology provides important information for diagnostic, prognostic, and even therapeutic applications. Several S100 proteins have been proposed to play important roles in tumor progression and are recognized as potential tumor markers. To substantiate these limited earlier findings, we screened hundreds of tumor specimens from patients of eight different tumor types using tissue microarrays. The results validated the expression of S100A4, S100A6, and S100B in specific tumor types. A significant S100A2 expression was observed in lymphoma biopsies, which implies a possible link between this S100 protein and lymphoma development. In contrast, S100A5 and S100A12 were not significantly expressed in any of the tumor tissues tested. Interestingly, expression of RAGE (receptor for advanced glycation end products) was found in breast and lung tumor tissues where abundant S100A4 and S100A6 expression was also observed. This suggests a possible role of RAGE-mediated signal transduction in the development of these particular cancers.     

Speed-mapping quantitative trait loci using microarrays

We developed a rapid, economical method for high-resolution quantitative trait locus (QTL) mapping using microarrays for selective genotyping of pooled DNA samples. We generated 21,207 F2 flies from two inbred Drosophila melanogaster strains with known QTLs affecting lifespan, and hybridized DNA pools of young and old flies to microarrays. We used changes of gene frequency of 2,326 single-feature polymorphisms (SFPs) to map previously identified and additional QTLs affecting lifespan.     

Probing lipid-protein interactions using lipid microarrays

Lipids are central to the regulation and control of several cellular functions. They form many of the important structural features of cells, and are critical members of cellular signal transduction pathways. Cellular dysfunction is often caused by errors in lipid signaling; therefore, the proteins that interact with, synthesize or metabolize the lipids are potential therapeutic targets. Characterizing the contingent of cellular lipids and their abundance and how this is associated with disease will facilitate understanding how to intervene to correct diseases caused by dysfunctional lipid signaling. Since lipid-signaling networks involve several classes of proteins it is essential to determine the identity and role of these proteins in order to understand the networks. These proteins may be receptors, effectors, transporters or enzymes. We present tools, specifically, a lipid microarray platform, to uncover lipid-binding effector proteins that function in lipid signaling pathways. Lipid microarrays will allow researchers to obtain a comparable fingerprint of the proteins from a cell or tissue that bind to lipids, and also enable the identification of functionally important lipid-binding proteins. By applying a systematic approach to the quantification of lipid-protein interactions, lipid microarrays will provide an integrated knowledge base for the human lipidome. These tools have the potential to identify and validate targets to improve personalized medicine and health.     

Comparing whole genomes using DNA microarrays

The rapid accumulation of complete genomic sequences offers the opportunity to carry out an analysis of inter- and intra-individual genome variation within a species on a routine basis. Sequencing whole genomes requires resources that are currently beyond those of a single laboratory and therefore it is not a practical approach for resequencing hundreds of individual genomes. DNA microarrays present an alternative way to study differences between closely related genomes. Advances in microarray-based approaches have enabled the main forms of genomic variation (amplifications, deletions, insertions, rearrangements and base-pair changes) to be detected using techniques that are readily performed in individual laboratories using simple experimental approaches.     

糖芯片技术在肿瘤研究中的应用

糖基化修饰是蛋白质常见的翻译后修饰之一,通过与糖结合蛋白如凝集素、抗体等相互作用调节肿瘤细胞侵袭、转移的能力及肿瘤异质性。通过化学合成法、化学-酶合成法或释放天然聚糖构建的糖芯片是分析聚糖与糖结合蛋白相互作用的重要工具。文中综述了常见的点制糖芯片的技术及糖芯片在癌症疫苗、单克隆抗体及诊断标志物中的广泛运用。由于肿瘤发生的各个环节都伴随着聚糖结构的改变,利用糖芯片探究肿瘤细胞特异表达的聚糖所参与的生理病理过程具有重大意义。