Biomedical subjects

Carolyn J M Best

Publications and source records attributed to Carolyn J M Best.

12 recordsLinked to original sources

PubMed2005

Molecular alterations in primary prostate cancer after androgen ablation therapy.

PURPOSE: After an initial response to androgen ablation, most prostate tumors recur, ultimately progressing to highly aggressive androgen-independent cancer. The molecular mechanisms underlying progression are not well known in part due to the rarity of androgen-independent samples from primary and metastatic sites. EXPERIMENTAL DESIGN: We compared the gene expression profiles of 10 androgen-independent primary prostate tumor biopsies with 10 primary, untreated androgen-dependent tumors. Samples were laser capture microdissected, the RNA was amplified, and gene expression was assessed using Affymetrix Human Genome U133A GeneChip. Differential expression was examined with principal component analysis, hierarchical clustering, and Student's t testing. Analysis of gene ontology was done with Expression Analysis Systematic Explorer and gene expression data were integrated with genomic alterations with Differential Gene Locus Mapping. RESULTS: Unsupervised principal component analysis showed that the androgen-dependent and androgen-independent tumors segregated from one another. After filtering the data, 239 differentially expressed genes were identified. Two main gene ontologies were found discordant between androgen-independent and androgen-dependent tumors: macromolecule biosynthesis was down-regulated and cell adhesion was up-regulated in androgen-independent tumors. Other differentially expressed genes were related to interleukin-6 signaling as well as angiogenesis, cell adhesion, apoptosis, oxidative stress, and hormone response. The Differential Gene Locus Mapping analysis identified nine regions of potential chromosomal deletion in the androgen-independent tumors, including 1p36, 3p21, 6p21, 8p21, 11p15, 11q12, 12q23, 16q12, and 16q21. CONCLUSIONS: Taken together, these data identify several unique characteristics of androgen-independent prostate cancer that may hold potential for the development of targeted therapeutic intervention.

Aged↗

PubMed2005

Optimal molecular profiling of tissue and tissue components: defining the best processing and microdissection methods for biomedical applications.

Isolation of well-preserved pure cell populations is a prerequisite for sound studies of the molecular basis of any tissue-based biological phenomenon. This article reviews current methods for obtaining anatomically specific signals from molecules isolated from tissues, a basic requirement for productive linking of phenotype and genotype. The quality of samples isolated from tissue and used for molecular analysis is often glossed over or omitted from publications, making interpretation and replication of data difficult or impossible. Fortunately, recently developed techniques allow life scientists to better document and control the quality of samples used for a given assay, creating a foundation for improvement in this area. Tissue processing for molecular studies usually involves some or all of the following steps: tissue collection, gross dissection/identification, fixation, processing/embedding, storage/archiving, sectioning, staining, microdissection/annotation, and pure analyte labeling/identification and quantification. We provide a detailed comparison of some current tissue microdissection technologies, and provide detailed example protocols for tissue component handling upstream and downstream from microdissection. We also discuss some of the physical and chemical issues related to optimal tissue processing, and include methods specific to cytology specimens. We encourage each laboratory to use these as a starting point for optimization of their overall process of moving from collected tissue to high quality, appropriately anatomically tagged scientific results. In optimized protocols is a source of inefficiency in current life science research. Improvement in this area will significantly increase life science quality and productivity. The article is divided into introduction, materials, protocols, and notes sections. Because many protocols are covered in each of these sections, information relating to a single protocol is not contiguous. To get the greatest benefit from this article, readers are advised to read through the entire article first, identify protocols appropriate to their laboratory for each step in their workflow, and then reread entries in each section pertaining to each of these single protocols.

Biomarkers↗

PubMed2005

Optimal molecular profiling of tissue and tissue components: defining the best processing and microdissection methods for biomedical applications.

Isolation of well-preserved pure cell populations is a prerequisite for sound studies of the molecular basis of pancreatic malignancy and other biological phenomena. This chapter reviews current methods for obtaining anatomically specific signals from molecules isolated from tissues, a basic requirement for productive linking of phenotype and genotype. The quality of samples isolated from tissue and used for molecular analysis is often glossed-over or omitted from publications, making interpretation and replication of data difficult or impossible. Fortunately, recently developed techniques allow life scientists to better document and control the quality of samples used for a given assay, creating a foundation for improvement in this area. Tissue processing for molecular studies usually involves some or all of the following steps: tissue collection, gross dissection/identification, fixation, processing/embedding, storage/archiving, sectioning, staining, microdissection/annotation, and pure analyte labeling/identification. High-quality tissue microdissection does not necessarily mean high-quality samples to analyze. The quality of biomaterials obtained for analysis is highly dependent on steps upstream and downstream from tissue microdissection. We provide protocols for each of these steps, and encourage you to improve upon these. It is worth the effort of every laboratory to optimize and document its technique at each stage of the process, and we provide a starting point for those willing to spend the time to optimize. In our view, poor documentation of tissue and cell type of origin and the use of nonoptimized protocols is a source of inefficiency in current life science research. Even incremental improvement in this area will increase productivity significantly.

Coloring Agents↗

PubMed2004

Comparison of snap freezing versus ethanol fixation for gene expression profiling of tissue specimens.

Frozen tissue specimens are the gold standard for molecular analysis. However, snap freezing presents several challenges regarding collection and storage of tissue, and preservation of histological detail. We evaluate an alternative preservation method, ethanol fixation followed by paraffin embedding, by analyzing expression profiles of microdissected cells on Affymetrix oligonucleotide arrays of three matched benign prostatic hyperplasia (BPH) and tumor samples processed with each preservation method. Frozen samples generated an average present call of 26% of the probe sets, compared to 4.5% in ethanol-paraffin samples. Eighty-eight percent of the probe sets called present in the ethanol-paraffin samples were also present in the frozen specimens. Comparing ethanol-paraffin BPH to tumor, 52 probe sets showed a twofold differential expression or higher in at least two cases, 23 of which were also differentially expressed in at least one frozen case. Despite a significant drop in the number of transcripts detectable, the data suggests that the obtainable information in ethanol-fixed samples may be useful for molecular profiling where frozen tissue is not available. However, ethanol fixation and paraffin embedding of tissue specimens is not optimal for high-throughput mRNA expression analysis. Improved methods for transcript profiling of archival samples, and/or tissue processing are still required.

Ethanol↗

PubMed2004

Expression microdissection: operator-independent retrieval of cells for molecular profiling.

Tissue microdissection is an important method for the study of disease states. However, it is difficult to perform high-throughput molecular analysis with current techniques. We describe here a prototype version of a novel technique (expression microdissection) that allows for the procurement of desired cells via molecular targeting. Expression microdissection (xMD) offers significant advantages over available methods, including an increase in dissection speed of several orders of magnitude. xMD may become a valuable tool for investigators studying cancer or other disease states in patient specimens and animal models.

Animals↗

PubMed2003

Molecular differentiation of high- and moderate-grade human prostate cancer by cDNA microarray analysis.

The prognosis of men with moderate-grade prostate cancer is uncertain. At present, there are few if any reliable molecular markers that can distinguish moderate-grade tumors from those that behave more aggressively. To better understand the molecular basis of human prostate cancer and potentially provide information toward more accurate prognosis, we measured and analyzed gene expression profiles of 13 high- and moderate-grade human prostate tumors using cDNA microarrays. The expression of 136 genes was observed to differ significantly (P < 0.001) between normal prostate and tumors using one-sample t testing and Wilcoxon ranking. Hierarchical clustering of genes demonstrated a relatively similar pattern of differential expression across the tumors. However, importantly, permutation t tests (two-tailed P < 0.001) revealed 21 genes whose expression profiles segregated moderate- and high-grade tumors from each other, which was significantly (P < 0.03) greater than what was expected by chance. These results were compared in silico with prostate cancer profiling efforts performed by other groups, including a meta-analysis of four data sets, which validated many of the dysregulated genes. We suggest that these data provide insight into the molecular nature of clinically aggressive prostate cancer.

Adenocarcinoma↗

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

Alterations in gene expression profiles during prostate cancer progression: functional correlations to tumorigenicity and down-regulation of selenoprotein-P in mouse and human tumors.

To identify molecular changes that occur during prostate tumor progression, we have characterized a series of prostate cancer cell lines isolated at different stages of tumorigenesis from C3(1)/Tag transgenic mice. Cell lines derived from low- and high-grade prostatic intraepithelial neoplasia, invasive carcinoma, and a lung metastasis exhibited significant differences in cell growth, tumorigenicity, invasiveness, and angiogenesis. cDNA microarray analysis of 8700 features revealed correlations between the tumorigenicity of the C3(1)/Tag-Pr cells and changes in the expression levels of genes regulating cell growth, angiogenesis, and invasion. Many changes observed in transcriptional regulation in this in vitro system are similar to those reported for human prostate cancer, as well as other types of human tumors. This analysis of expression patterns has also identified novel genes that may be involved in mechanisms of prostate oncogenesis or serve as potential biomarkers or therapeutic targets for prostate cancer. Examples include the L1-cell adhesion molecule, metastasis-associated gene (MTA-2), Rab-25, tumor-associated signal transducer-2 (Trop-2), and Selenoprotein-P, a gene that binds selenium and prevents oxidative stress. Many genes identified in the Pr-cell line model have been shown to be altered in human prostate cancer. The comprehensive microarray data provides a rational basis for using this model system for studies where alterations of specific genes or pathways are of particular interest. Quantitative real-time reverse transcription-PCR for Selenoprotein-P demonstrated a similar down-regulation of the transcript of this gene in a subset of human prostate tumors, mouse tumors, and prostate carcinoma cell lines. This work demonstrates that expression profiling in animal models may lead to the identification of novel genes involved in human prostate cancer biology.

Animals↗

PubMed2002

Proteomic analysis of human prostate cancer.

Proteomics is a promising approach in the identification of proteins and biochemical pathways involved in tumorigenesis. In an effort to discover such proteins and pathways that are deregulated in prostate tumorigenesis, cellular proteomes of matched normal prostate epithelial cells and high-grade prostate cancer cells were analyzed by tissue microdissection, two-dimensional electrophoresis, and mass spectrometry. Forty protein alterations were detected in the tumors; however, the majority of these changes were not shared among the 12 neoplasms. In contrast, parallel cDNA microarray analysis identified a number of common gene expression changes. The marked heterogeneity of the observed protein alterations may have significance with regard to tumor biology and research strategies for molecular profiling analyses of human prostate cancer.

Electrophoresis, Gel, Two-Dimensional↗

PubMed2002

Evaluation of non-formalin tissue fixation for molecular profiling studies.

Using a general strategy for evaluating clinical tissue specimens, we found that 70% ethanol fixation and paraffin embedding is a useful method for molecular profiling studies. Human prostate and kidney were used as test tissues. The protein content of the samples was analyzed by one-dimensional gel electrophoresis, immunoblot, two-dimensional gel electrophoresis, and layered expression scanning. In each case, the fixed and embedded tissues produced results similar to that obtained from snap-frozen specimens, although the protein quantity was somewhat decreased. Recovery of mRNA was reduced in both quantity and quality in the ethanol-fixed samples, but was superior to that obtained from formalin-fixed samples and sufficient to perform reverse transcription polymerase chain reactions. Recovery of DNA from ethanol-fixed specimens was superior to formalin-fixed samples as determined by one-dimensional gel electrophoresis and polymerase chain reaction. In conclusion, specimens fixed in 70% ethanol and embedded in paraffin produce good histology and permit recovery of DNA, mRNA, and proteins sufficient for several downstream molecular analyses. Complete protocols and additional discussion of relevant issues are available on an accompanying website (http://cgap-mf.nih.gov/).

Actins↗

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↗

PubMedDate not supplied

Molecular profiling of cancer.

The objective of molecular profiling of cancer is to determine the differential expression of genes and proteins from human tissue in the progression from normal precursor tissue to preneoplastic tissue to cancer in order to discover diagnostic, prognostic, and therapeutic markers. With the development of high-throughput analytical techniques such as microarrays and 2-D PAGE as well as the development of tools for cell procurement from histological sections such as laser capture microdissection (LCM), it is now possible to perform molecular analyses on specific cell populations from tissue. Since recognition of specific cell populations is critical, there is a need to optimize fixation and embedding not only to improve preservation of biomolecules, but also to maintain excellent histology. We have shown that 70% ethanol fixation of prostate tissue improves the recovery of DNA, RNA, and proteins over routine formalin fixation and maintains histological quality comparable to formalin. There is also a need to develop new technologies in order to expand the range of tissue types that can be analyzed. The development and applications of Layered Expression Scanning (LES) for the molecular analysis of whole tissue sections are discussed.

DNA, Neoplasm↗