Biomedical subjects

Suzanne M Grindle

Publications and source records attributed to Suzanne M Grindle.

5 recordsLinked to original sources

PubMed2005

Functional analysis of human hematopoietic stem cell gene expression using zebrafish.

Although several reports have characterized the hematopoietic stem cell (HSC) transcriptome, the roles of HSC-specific genes in hematopoiesis remain elusive. To identify candidate regulators of HSC fate decisions, we compared the transcriptome of human umbilical cord blood and bone marrow (CD34+)(CD33-)(CD38-)Rho(lo)(c-kit+) cells, enriched for hematopoietic stem/progenitor cells with (CD34+)(CD33-)(CD38-)Rho(hi) cells, enriched in committed progenitors. We identified 277 differentially expressed transcripts conserved in these ontogenically distinct cell sources. We next performed a morpholino antisense oligonucleotide (MO)-based functional screen in zebrafish to determine the hematopoietic function of 61 genes that had no previously known function in HSC biology and for which a likely zebrafish ortholog could be identified. MO knock down of 14/61 (23%) of the differentially expressed transcripts resulted in hematopoietic defects in developing zebrafish embryos, as demonstrated by altered levels of circulating blood cells at 30 and 48 h postfertilization and subsequently confirmed by quantitative RT-PCR for erythroid-specific hbae1 and myeloid-specific lcp1 transcripts. Recapitulating the knockdown phenotype using a second MO of independent sequence, absence of the phenotype using a mismatched MO sequence, and rescue of the phenotype by cDNA-based overexpression of the targeted transcript for zebrafish spry4 confirmed the specificity of MO targeting in this system. Further characterization of the spry4-deficient zebrafish embryos demonstrated that hematopoietic defects were not due to more widespread defects in the mesodermal development, and therefore represented primary defects in HSC specification, proliferation, and/or differentiation. Overall, this high-throughput screen for the functional validation of differentially expressed genes using a zebrafish model of hematopoiesis represents a major step toward obtaining meaningful information from global gene profiling of HSCs.

ADP-ribosyl Cyclase 1↗

PubMed2004

Gene expression profiles in esophageal adenocarcinoma.

BACKGROUND: The incidence of esophageal adenocarcinoma (EAC) has risen dramatically in the last two decades. As with other malignancies, changes in gene expression play a key role in the development and progression of these tumors. METHODS: Microarray analysis was used to study gene expression of 12,000 genes in EAC specimens. Adenocarcinoma tissue samples (n = 10) and controls of normal stomach (n = 6) and esophageal (n = 7) mucosa were collected fresh, then rapidly frozen in liquid nitrogen. The messenger ribonucleic acid (mRNA) from the samples was isolated, reverse transcribed, and used to generate biotin-labeled mRNA fragments, which were hybridized to Affymetrix U95 gene chips (AME Bioscience, Norway) for analysis. Additional samples analyzed included tissue containing dysplastic Barrett's epithelium from three patients, metastatic lymph nodes from two patients with EAC, one squamous carcinoma, and two esophageal cancer cell lines. Samples were segregated into groups with similar patterns of gene expression using clustering algorithms and gene sets that differentiated tumors from normal tissue were generated. RESULTS: There were 150 genes that were fourfold up regulated and 183 genes that were fourfold down regulated in the esophageal adenocarcinoma specimens, as compared to normal esophageal mucosa tissue controls. Using paired specimens (n = 5) and the paired t-test (p Value of 0.05) as a filter, only 64 genes were fourfold up regulated and 110 were fourfold down regulated. These groups included cytoskeletal, cell adhesion, tumor suppressor, and signal transduction genes. Hierarchical clustering segregated the samples into the expected divisions. The esophageal cancer cell lines, OE19 and OE33, clustered separately from the EAC specimens. Extremely high gene expression levels of the ERBB2 gene, seen in the microarray analysis of the 2 cell lines, correlated with amplification of the gene determined by Southern blotting. CONCLUSIONS: Gene expression patterns from a small subset of genes distinguish EAC specimens from normal controls. This technique can rapidly identify genes for targeted chemotherapeutic approaches to cancer treatment.

Adenocarcinoma↗

PubMed2004

Differential gene expression in ovarian carcinoma: identification of potential biomarkers.

Ovarian cancer remains the fifth leading cause of cancer death for women in the United States. In this study, the gene expression of 20 ovarian carcinomas, 17 ovarian carcinomas metastatic to the omentum, and 50 normal ovaries was determined by Gene Logic Inc. using Affymetrix GeneChip HU_95 arrays containing approximately 12,000 known genes. Differences in gene expression were quantified as fold changes in gene expression in ovarian carcinomas compared to normal ovaries and ovarian carcinoma metastases. Genes up-regulated in ovarian carcinoma tissue samples compared to more than 300 other normal and diseased tissue samples were identified. Seven genes were selected for further screening by immunohistochemistry to determine the presence and localization of the proteins. These seven genes were: the beta8 integrin subunit, bone morphogenetic protein-7, claudin-4, collagen type IX alpha2, cellular retinoic acid binding protein-1, forkhead box J1, and S100 calcium-binding protein A1. Statistical analyses showed that the beta8 integrin subunit, claudin-4, and S100A1 provided the best distinction between ovarian carcinoma and normal ovary tissues, and may serve as the best candidate tumor markers among the seven genes studied. These results suggest that further exploration into other up-regulated genes may identify novel diagnostic, therapeutic, and/or prognostic biomarkers in ovarian carcinoma.

Adenocarcinoma, Papillary↗

PubMed2003

Cell membrane glycosylation mediates the adhesion, migration, and invasion of ovarian carcinoma cells.

We have previously shown that ovarian carcinoma cell adhesion to mesothelial cell monolayers and migration toward fibronectin, type IV collagen, and laminin is partially mediated by CD44, a proteoglycan known to affect the functional abilities of tumor cells. The purpose of this study was to determine the role of cell membrane glycosylation in the metastatic abilities of ovarian carcinoma cells. NIH:OVCAR5 cells were treated with glycosidases to remove carbohydrate moieties from molecules on the cells' surface. The ability of the treated cells to adhere to extracellular matrix components or mesothelial cell monolayers, migrate toward extracellular matrix proteins, and invade through Matrigel was assessed. We observed that the loss of different carbohydrate moieties resulted in altered ovarian carcinoma cell adhesion, migration, and/or invasion toward extracellular matrix components or mesothelial cell monolayers. Gene array analysis of NIH:OVCAR5 cells revealed the expression of several proteoglycans, including syndecan 4, decorin, and perlecan. In tissue samples obtained from patients, altered proteoglycan gene expression was observed in primary ovarian carcinoma tumors and secondary metastases, compared to normal ovaries. Taken together, these results suggest that ovarian carcinoma cell proteoglycans affect the cells' ability to adhere, migrate, and invade toward extracellular matrix components and mesothelial cell monolayers. Thus, the carbohydrate modifications of several proteoglycans may mediate the formation and spread of secondary tumor growth in ovarian carcinoma.

Cell Adhesion↗

PubMed2003

Establishment of an in vitro assay to measure the invasion of ovarian carcinoma cells through mesothelial cell monolayers.

Ovarian carcinoma is the leading cause of gynecological cancer deaths in the United States. Secondary tumor growths form by tumor cell invasion through the mesothelial lining of the peritoneal cavity and peritoneal organs. To study this interaction, we developed a dye-based in vitro model system in which mesothelial cells were grown as confluent monolayers, permeabilized, and then co-cultured with ovarian carcinoma cells for up to seven days. The mesothelial cells were then stained with trypan blue dye, which enabled the visualization of ovarian carcinoma cell invasion through the monolayers of mesothelial cells. Ovarian carcinoma cell invasion was inhibited for up to 7 days by the addition of GRGDSP peptides, a blocking monoclonal antibody against the beta1 integrin subunit, or blocking monoclonal antibodies against matrix metalloproteinases 2 and 9. Cell invasion was also inhibited by hyaluronan and GM6001, a chemical inhibitor of matrix metalloproteinases. Differential gene expression of matrix metalloproteinases, tissue inhibitors of matrix metalloproteinases, and disintegrins were observed in primary ovarian carcinoma tumors and secondary metastases, compared to normal ovaries. Taken together, these results suggest that complex interactions between integrins, disintegrins, matrix metalloproteinases, and tissue inhibitors of matrix metalloproteinases may mediate ovarian carcinoma cell invasion, and that the dye-based assay described herein is a suitable model system for its study.

Adenocarcinoma↗