Identification of differentially expressed genes in human uterine leiomyomas using differential display

INTRODUCTIONUterine leiomyomas (ULs) have been consideredto be of uniceIIular origin[l1. It is one of the mostcommon benign tumors, occurring in 20% to 30% ofwomen[2], accounting for significant morbidity andusually need major surgery[3] which might causesome side effects afterwards[4]. Therefore, to de-velop certain drug treatments instead has been thehope of these patients for a long time. Using alter-native approaches fOr studying patients sufferingfrom leiomyoma in various ethnic gr…     

Identification by differential display of a hypertonicity-inducible inward rectifier potassium channel highly expressed in chloride cells

By using differential mRNA display to monitor the molecular alterations associated with adaptation of euryhaline eels to different salinities, we identified a cDNA fragment strongly induced in seawater eel gills. Cloning of a full-length cDNA and its expression in COS-7 cells indicated that the clone codes for an inward rectifier K+ channel (eKir) of 372 amino acid residues, which has two transmembrane segments and a typical pore-forming region (H5). Only low sequence similarities are present, except the H5 region, compared with other members of the inward rectifier K+ channel family (Kir). Consistent with this divergence in the amino acid sequence, a phylogenetic analysis indicated early divergence and independent evolution of eKir from other members; it is only distantly related to the Kir5.0 subfamily members. RNase protection analysis showed that eKir is highly expressed in the seawater eel gill, kidney, and posterior intestine but very weakly in freshwater eels. Immunohistochemistry of gill sections revealed dense localization of eKir in the chloride cells. Immunoelectron microscopy indicated that eKir is mainly present in the microtubular system in the chloride cell. This location and its salt-inducible nature suggest that the eKir channel cloned here is a novel member of the Kir5.0 subfamily of the Kir family and is implicated in osmoregulation.     

Identification of mRNAs differentially expressed in quiescence or in late G1 phase of the cell cycle in human breast cancer cells by using the differential display method.

BACKGROUND: The decision for a cell to enter the DNA synthesis (S) phase of the cell cycle or to arrest in quiescence is likely to be determined by genes expressed in the late G1 phase, at the restriction point. Loss of restriction point control is associated with malignant cellular transformation and cancer. For this reason, identifying genes that are differentially expressed in late G1 phase versus quiescence is important for understanding the molecular basis of normal and malignant growth. MATERIALS AND METHODS: The differential display (DD) method detects mRNA species that are different between sets of mammalian cells, allowing their recovery and cloning of the corresponding cDNAs. Using this technique, we compared mRNAs from synchronized human breast cancer cells (21 PT) in quiescence and in late G1. RESULTS: Six mRNAs differentially expressed in late G1 or in quiescence were identified. One mRNA expressed 10 hr after serum induction showed 99% homology to a peptide transporter involved in antigen presentation of the class I major histocompatibility complex (TAP-1) mRNA. Another mRNA expressed specifically in quiescence and down-regulated 2 hr following serum induction showed 98% homology to human NADP+ -dependent cytoplasmic malic enzyme (EC1.1.1.40) mRNA, which is an important enzyme in fatty acid synthesis and lipogenesis. Three others showed high homology to different mRNAs in the GeneBank, corresponding to genes having unknown functions. Finally, one mRNA revealed no significant homology to known genes in the GeneBank. CONCLUSIONS: We conclude that DD is an efficient and powerful method for the identification of growth-related genes which may have a role in cancer development.     

Identification of a glialblastoma cell differentiation factor-related gene mRNA in human microvascular endothelial cells

Vascular endothelial cells (VEC) transduce mitogenic and chemoattractant signals in response to erythropoietin (Epo). An analysis of changes in gene expression in VEC would be helpful to understanding the molecular nature of mitogenic signals. An effective method for analysis of gene expression is through differential display. Using this approach, we obtained from Epo-treated human microvascular endothelial cells (HMVEC) a cDNA fragment with characteristics of the 3'end of mRNA. Using the cDNA fragment, we then isolated a full-length clone from a HMVEC cDNA library. The cDNA of interest encodes a protein consisting of 404 amino acids with a carboxy-terminal end sequence identical to glialblastoma cell differentiation factor-related protein (GBDR1). Northern blot analysis showed that GBDR1 mRNA was ubiquitously expressed in human tissues. In Southern blot analysis, GBDR1 cDNA identified a single gene on chromosome 9. Since analysis of the amino acid sequence revealed several putative phosphorylation sites for different protein kinases, the GBDR1 protein was expressed and purified from bacterial extracts and, as predicted, casein kinase II phosphorylated GBDR1 in vitro. Immunofluorescence and biochemical data revealed that the GBDR1 protein is not entirely localized in the cytosolic fraction, suggesting that it may interact with another protein(s). These findings demonstrate that GBDR1 is an intracellular signaling molecule that may play a role in the regulation of endothelial cell growth.     

Screening differentially expressed cDNA clones obtained by differential display using amplified RNA.

The major obstacle of differential display is not the technique itself but rather the post-differential display issueof discriminating between false positives and the truly differentially expressed mRNAs. This process is arduous and requires large amounts of RNA. We present and validate a method which allows one to screen putative positives from differential display analysis using only micrograms of total RNA. More importantly, we demonstrate that cDNA probes generated from amplified RNA are representative of the starting mRNA population and can be used for differential screening of mRNA species at a detectable limit of sensitivity of>/=1/40 000.     

Regulation of sterol transport in human microvascular endothelial cells

In cultured human dermal microvessel endothelial cells, the rate of efflux (about twofold greater than for fibroblasts under equivalent conditions) was coupled to an equivalent high rate of sterol net transport from the cells to the medium. This net transport was linked with esterification via lecithin:cholesterol acyltransferase. Since the use of free sterol by plasma transferase is constant, such increased net transport indicates that endothelial cells are highly efficient, in competition with plasma lipoproteins, in supplying free sterol for esterification. These results indicate the marked ability of endothelial cells to regulate and maintain their sterol balance in the face of high sterol levels to which these cells are uniquely exposed in human plasma.     

Spontaneous immortalization of human dermal microvascular endothelial cells

AIM: To establish and characterize a spontaneously immortalized human dermal microvascular endothelial cell line, iHDME1.METHODS: We developed a spontaneous immortalization method. This approach is based on the application of optimized culture media and culture conditions without addition of any exogenous oncogenes or carcinogens. Using this approach, we have successfully established a microvascular endothelial cell line, iHDME1, from primary human dermal microvascular endothelial cells. iHDME1 cells have been maintained in culture dishes for more than 50 passages over a period of 6 mo. Using a GFP expressing retrovirus, we generated a GFP-stable cell line (iHDME1-GFP).RESULTS: iHDME1 retain endothelial morphology and uniformly express endothelial markers such as VEGF receptor 2 and VE-cadherin but not α-smooth muscle actin (α-SM-actin) and cytokeratin 18, markers for smooth muscle cells and epithelial cells respectively. These cells retain endothelial properties, migrate in response to VEGF stimulation and form 3-D vascular structures in Matrigel, similar to the parental cells. There is no significant difference in cell cycle profile between the parental cells and iHDME1 cells. Further analysis indicates enhanced stemness in iHDME1 cells compared to parental cells. iHDME1 cells display elevated expression of CD133 and hTERT.CONCLUSION: iHDME1 cells will be a valuable source for studying angiogenesis.     

Proteomic analysis of the proteins expressed by hydrogen peroxide treated cultured human dermal microvascular endothelial cells

Reactive oxygen species (ROS) have been traditionally regarded as toxic by-products of aerobic metabolism. However, ROS also act as intracellular signaling molecules and can mediate phenotypes in vascular endothelial cells, which may be physiological or pathological in nature. To clarify the molecular mechanisms of ROS signaling, we examined hydrogen peroxide (H(2)O(2))-responsive proteins in cultured human dermal microvascular endothelial cells (HMVEC) using proteomic tools. Protein expression in HMVEC was studied after they had been exposed to low- and high-levels of H(2)O(2) for various times, and intracellular ROS production was examined by flow cytometer and UV spectrophotometer. Proteins obtained from dose- and time-dependent series were separated by two-dimensional gel electrophoresis and tentatively identified by matrix-assisted laser desorption-time of flight mass spectrometry, by matching the tryptic mass maps obtained with entries in the NCBI and Swiss-Prot protein sequence database. At least 163 proteins were changed by H(2)O(2), and 60 proteins were identified. Oxidative stress triggered dramatic change in the expression of proteins in primary microvessel endothelial cells, and their mapping to cellular process provided a view of the ubiquitous cellular changes elicited by H(2)O(2). These results could provide a framework for the understanding of the mechanisms of cellular redox homeostasis and H(2)O(2) metabolism in microendothelium environment in various biological processes as well as pathological conditions.     

Identification of a hybrid-specific expressed gene encoding novel RNA-binding protein in wheat seedling leaves using differential display of mRNA

A hybrid-specific expressed cDNA fragment, designated as AG5, has been identified in wheat seedling leaves using differential mRNA display. AG5 contains an open reading frame (ORF) encoding 183 amino acid residues. Comparison with amino acid sequences in GenBank revealed that the AG5 protein is homologous to a group of Gly-rich proteins with consensus sequence-type RNA-binding domains (CS-RBD). Structural analysis showed that AG5 protein contains five motifs, including a consensus sequence-type RNA-binding domain near its N-terminus, arginine/aspartic acid repeats and a Gly-rich region in its center, a Cys-X₂-Cys-X₄-His-X₄-Cys (CCHC) zinc finger motif in the Gly-rich region, and TrySer₂ArgAsp₂Arg repeats towards its C-terminus. Of all previously described RNA-binding proteins, only RZ-1 from tobacco has a similar structure to the AG5 protein, but RZ-1 lacks a TrySer2ArgAsp2Arg repeat motif, indicating that the two proteins may belong to a family of closely related proteins in plants. The possible role of AG5 and its relation to wheat heterosis are discussed. Received: 21 November 1999 / Accepted: 20 March 2000     

Cancer cells regulate biomechanical properties of human microvascular endothelial cells

Metastasis is a key event of malignant tumor progression. The capability to metastasize depends on the ability of the cancer cell to migrate into connective tissue, adhere, and possibly transmigrate through the endothelium. Previously we reported that the endothelium does not generally act as barrier for cancer cells to migrate in three-dimensional extracellular matrices (3D-ECMs). Instead, the endothelium acts as an enhancer or a promoter for the invasiveness of certain cancer cells. How invasive cancer cells diminish the endothelial barrier function still remains elusive. Therefore, this study investigates whether invasive cancer cells can decrease the endothelial barrier function through alterations of endothelial biomechanical properties. To address this, MDA-MB-231 breast cancer cells were used that invade deeper and more numerous into 3D-ECMs when co-cultured with microvascular endothelial cells. Using magnetic tweezer measurements, MDA-MB-231 cells were found to alter the mechanical properties of endothelial cells by reducing endothelial cell stiffness. Using spontaneous bead diffusion, actin cytoskeletal remodeling dynamics were shown to be increased in endothelial cells co-cultured with MDA-MB-231 cells compared with mono-cultured endothelial cells. In addition, knockdown of the α5 integrin subunit in highly transmigrating α5β1(high) cells derived from breast, bladder, and kidney cancer cells abolished the endothelial invasion-enhancing effect comparable with the inhibition of myosin light chain kinase. These results indicate that the endothelial invasion-enhancing effect is α5β1 integrin-dependent. Moreover, inhibition of Rac-1, Rho kinase, MEK kinase, and PI3K reduced the endothelial invasion-enhancing effect, indicating that signaling via small GTPases may play a role in the endothelial facilitated increased invasiveness of cancer cells. In conclusion, decreased stiffness and increased cytoskeletal remodeling dynamics of endothelial cells may account for the breakdown of endothelial barrier function, suggesting that biomechanical alterations are sufficient to facilitate the transmigration and invasion of invasive cancer cells into 3D-ECMs.