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PubMed1998

Reciprocal subtraction differential RNA display: an efficient and rapid procedure for isolating differentially expressed gene sequences.

A reciprocal subtraction differential RNA display (RSDD) approach has been developed that permits the rapid and efficient identification and cloning of both abundant and rare differentially expressed genes. RSDD comprises reciprocal subtraction of cDNA libraries followed by differential RNA display. The RSDD strategy was applied to analyze the gene expression alterations resulting during cancer progression as adenovirus-transformed rodent cells developed an aggressive transformed state, as documented by elevated anchorage-independence and enhanced in vivo oncogenesis in nude mice. This approach resulted in the identification and cloning of both known and a high proportion (>65%) of unknown sequences, including cDNAs displaying elevated expression as a function of progression (progression-elevated gene) and cDNAs displaying suppressed expression as a function of progression (progression-suppressed gene). Sixteen differentially expressed genes, including five unknown progression-elevated genes and six unknown progression-suppressed genes, have been characterized. The RSDD scheme should find wide application for the effective detection and isolation of differentially expressed genes.

Animals↗
PubMed2005

Systematic identification and molecular characterization of genes differentially expressed in breast and ovarian cancer.

The identification of novel disease-associated genes in gynaecological tumours has important implications for understanding the process of tumourigenesis and the development of novel treatment regimens. cDNA libraries from disease tissues may represent a valuable source to identify such genes. Recently, a bio-informatic procedure based on an 'electronic Northern' approach was established to screen expressed sequence tag (EST) libraries for genes differentially expressed in tumour and normal tissues, and identified 450 candidate genes differentially expressed in breast and ovarian cancer. In this report, the validation of an initial set of 40 candidate genes, which were selected due to their localization in chromosomal regions frequently altered in gynaecological tumours, is described. Differential expression of 29 of these genes, including three uncharacterized novel genes, was confirmed by applying cancer profiling arrays with 106 matched pairs of tumour/normal cDNAs and quantitative reverse transcription-polymerase chain reaction (RT-PCR) on 60 clinical specimens. The majority of these differentially expressed genes have not been described previously in the context of breast and ovarian cancer, and may constitute novel diagnostic markers for these tumour entities.

Breast Neoplasms↗
PubMed2000

A comparison of aorta and vena cava medial message expression by cDNA array analysis identifies a set of 68 consistently differentially expressed genes, all in aortic media.

We performed a systematic analysis of gene expression in arteries and veins by comparing message profiles of macaque aorta and vena cava media using a cDNA array containing 4048 known human genes, approximately 35% of currently named human genes (approximately 11,000). The data show extensive differences in RNA expression in artery versus vein media. Sixty-eight genes had consistent elevation in message expression by the aorta, but none were elevated in the vena cava. The most differentially expressed gene was regulator of G-protein signaling (RGS) 5, at an expression ratio of 46.5+/-12.6 (mean+/-SEM). The data set also contained 2 genes already known to be expressed in the aorta, elastin at 5.0+/-1.4, and the aortic preferentially expressed gene 1 (APEG-1) at 2.3+/-0.6. We chose to analyze RGS5 expression further because of its high level of differential expression in the aorta. Levels of RGS5 mRNA were confirmed by Northern analysis and in situ hybridization. A human tissue RNA dot blot showed that RGS5 message is highest in aorta, followed by small intestine, stomach, and then heart. Northern analysis confirmed that RGS5 expression in human aorta is higher than in any region of the heart. RGS5 is a G-protein signaling regulator of unknown specificity most homologous to RGS4, an inhibitory regulator of pressure-induced cardiac hypertrophy. The expression pattern of the 68 differential genes as a whole is a start toward identifying the molecular phenotypes of arteries and veins on a systematic basis.

Adult↗
PubMed1997

Differential expression of Rab3 isoforms during differentiation of pancreatic acinar cell line AR42J.

Evidence is accumulating that low molecular weight GTPases of the Rab3 subfamily regulate exocytosis in secretory cells. The different isoforms of Rab3 (Rab3AD) probably have opposite roles in secretory processes. In the present study we have investigated differentiation-dependent expression of Rab3 isoforms in the pancreatic acinar carcinoma cell line AR42J. The data show that AR42J cells express all four different isoforms of Rab3. Dexamethasone treatment, which increases the number of secretory granules and secretagogue-induced exocytosis several-fold, was accompanied by down-regulation of Rab3A, Rab3C, and Rab3D mRNA, whereas Rab3B mRNA and Rab3 protein were found up-regulated at the mRNA and protein level, suggesting that Rab3B might be involved in the secretory function of AR42J cells.

Amino Acid Sequence↗
PubMed2006

The expression levels and the differential expression of transforming growth factor-beta receptors in dermatofibroma and dermatofibrosarcoma protuberans.

BACKGROUND: Dermatofibroma (DF) and dermatofibrosarcoma protuberans (DFSP) are benign and intermediate malignant fibrotic dermal tumours, respectively. The contribution of transforming growth factor (TGF)-beta has been implicated in the progression of sclerosis in fibrotic diseases. OBJECTIVES: To investigate the expression of TGF-beta receptors in these fibrotic tumours. METHODS: We examined the expression levels of TGF-beta type I and type II receptors (TGFbeta-RI and TGFbeta-RII) in DF and DFSP using in situ hybridization and immunohistochemical analysis. We also examined the expression of TGF-beta1 and collagen type I using immunohistochemical analysis. RESULTS: We detected strong expression of TGFbeta-RI and TGFbeta-RII on epidermis and epidermal appendages, moderate expression in vascular endothelial cells, smooth muscle cells and neural tissues, and weak expression in fibroblasts in normal skin sections. The expression levels of TGFbeta-RI and TGFbeta-RII were elevated in the tissue sections of DF in comparison with normal dermal sections using in situ hybridization and immunohistochemical staining. Furthermore, the expression of TGFbeta-RI and TGFbeta-RII was strong in spindle-shaped cells around DF. The expression of TGFbeta-RI and TGFbeta-RII was decreased in DFSP in comparison with DF, and their expression was found to be homogeneous in each DFSP tumour cell. The staining for TGF-beta1 was found prominently on matrix and spindle-shaped tumour cells of DF, and peripheral regions of DFSP. Weak expression of TGF-beta1 was found on normal skin or tumour cells in the central part of DFSP. Type I collagen expression was found on spindle-shaped tumour cells in DF, but not in tumour cells of DFSP. CONCLUSIONS: These results suggest the possibility that TGF-beta signalling may contribute to the fibrosis around DF, and that TGF-beta receptors may play important roles in TGF-beta signalling. The expression patterns of TGFbeta-RI and TGFbeta-RII may be helpful in distinguishing these diseases.

Activin Receptors, Type I↗
PubMed2006

Gene expression profiling separates chromophobe renal cell carcinoma from oncocytoma and identifies vesicular transport and cell junction proteins as differentially expressed genes.

PURPOSE: To compare gene expression profiles of chromophobe renal cell carcinoma (RCC) and benign oncocytoma, aiming at identifying differentially expressed genes. EXPERIMENTAL DESIGN: Nine cases each of chromophobe RCC and oncocytoma were analyzed by oligonucleotide microarray. Candidate genes that showed consistent differential expression were validated by reverse transcription-PCR using 25 fresh-frozen and 15 formalin-fixed, paraffin-embedded tumor samples. Immunohistochemical analysis was also done for two selected gene products, claudin 8 and MAL2. RESULTS: Unsupervised hierarchical clustering separated the chromophobe RCC and oncocytoma into two distinct groups. By a combination of data analysis approaches, we identified 11 candidate genes showing consistent differential expression between chromophobe RCC and oncocytoma. Five of these genes, AP1M2, MAL2, PROM2, PRSS8, and FLJ20171, were shown to effectively separate these two tumor groups by quantitative reverse transcription-PCR using fresh tissue samples, with similar trends seen on formalin-fixed tissues. Immunohistochemical analysis revealed selective expression of MAL2 and claudin 8 in distal renal tubules, with MAL2 antibody showing differential expression between chromophobe RCC and oncocytoma. Functional analyses suggest that genes encoding tight junction proteins and vesicular membrane trafficking proteins, normally expressed in distal nephrons, are retained in chromophobe RCC and lost or consistently down-regulated in oncocytoma, indicating that these two tumor types, believed to be both derived from distal tubules, are likely distinctive in their histogenesis. CONCLUSIONS: We showed that chromophobe RCC and oncocytoma are distinguishable by mRNA expression profiles and a panel of gene products potentially useful as diagnostic markers were identified.

Adaptor Protein Complex 1↗
PubMed2007

Differential expression profile prioritization of positional candidate glaucoma genes: the GLC1C locus.

OBJECTIVES: To develop and apply a model for prioritization of candidate glaucoma genes. METHODS: This Affymetrix GeneChip (Affymetrix, Santa Clara, Calif) study of gene expression in primary culture human trabecular meshwork cells uses a positional differential expression profile model for prioritization of candidate genes within the GLC1C genetic inclusion interval. RESULTS: Sixteen genes were expressed under all conditions within the GLC1C interval. TMEM22 was the only gene within the interval with differential expression in the same direction under both conditions tested. Two genes, ATP1B3 and COPB2, are of interest in the context of a protein-misfolding model for candidate selection. SLC25A36, PCCB, and FNDC6 are of lesser interest because of moderate expression and changes in expression. Transcription factor ZBTB38 emerges as an interesting candidate gene because of the overall expression level, differential expression, and function. CONCLUSIONS: Only 1 gene in the GLC1C interval fits our model for differential expression under multiple glaucoma risk conditions. The use of multiple prioritization models resulted in filtering 7 candidate genes of higher interest out of the 41 known genes in the region. CLINICAL RELEVANCE: This study identified a small subset of genes that are most likely to harbor mutations that cause glaucoma linked to GLC1C.

Adolescent↗
PubMed2006

Phenotypic and functional heterogeneity of GFAP-expressing cells in vitro: differential expression of LeX/CD15 by GFAP-expressing multipotent neural stem cells and non-neurogenic astrocytes.

Recent findings show that the predominant multipotent neural stem cells (NSCs) isolated from postnatal and adult mouse brain express glial fibrillary acid protein (GFAP), a protein commonly associated with astrocytes, and that primary astrocyte cultures can contain GFAP-expressing cells that act as multipotent NSCs when transferred to neurogenic conditions. The relationship of GFAP-expressing NSCs to GFAP-expressing astrocytes is unclear, but has important implications. We compared the phenotype and neurogenic potential of GFAP-expressing cells derived from different CNS regions and maintained in vitro under different conditions. Multiple labeling immunohistochemistry revealed that both primary astrocyte cultures and adherent neurogenic cultures derived from postnatal or adult periventricular tissue contained subpopulations of GFAP-expressing cells that co-expressed nestin and LeX/CD15, two molecules associated with NSCs. In contrast, GFAP-expressing cells in similar cultures prepared from adult cerebral cortex did not express detectable levels of LeX/CD15, and exhibited no neurogenic potential. Fluorescence-activated cell sorting (FACS) of both primary astrocyte cultures and adherent neurogenic cultures for LeX/CD15 showed that GFAP-expressing cells competent to act as multipotent NSCs were concentrated in the LeX-positive fraction. Using neurosphere assays and a transgenic ablation strategy, we confirmed that the predominant NSCs in primary astrocyte and adherent neurogenic cultures were GFAP-expressing cells. These findings demonstrate that GFAP-expressing cells derived from postnatal and adult forebrain are heterogeneous in both molecular phenotype and neurogenic potential in vitro, and that this heterogeneity exists before exposure to neurogenic conditions. The findings provide evidence that GFAP-expressing NSCs are phenotypically and functionally distinct from non-neurogenic astrocytes.

Animals↗
PubMed1990

Members of the RCK potassium channel family are differentially expressed in the rat nervous system.

mRNAs encoding four members of the RCK potassium channel family, named RCK1, RCK3, RCK4 and RCK5 have been analyzed by RNA blot hybridization experiments using specific RNA probes. Each probe recognizes a single mRNA species, their sizes ranging from approximately 4600 nucleotides up to approximately 11,000 nucleotides. The expression of RCK mRNAs as well as their developmental appearance in different regions of the central and peripheral rat nervous system has been investigated. The two most abundant RCK potassium channel mRNAs (RCK1 and RCK5) are predominantly expressed in the adult nervous system. RCK3 and RCK4 mRNAs are present throughout all developmental stages studied. The temporal and regional patterns observed are specific for each RCK potassium channel mRNA indicating that specific regulation of expression occurs. Differential mRNA expression might provide one mechanism for the generation of potassium channel diversity in vivo.

Animals↗
PubMed1995

A mitochondrial elongation factor-like protein is over-expressed in tumours and differentially expressed in normal tissues.

The tissue-specific expression of an antigen (P43) ubiquitously expressed at high levels in a variety of tumours of human and animal origin was investigated using a monoclonal antibody to P43. Whereas low amounts of P43 are expressed in the spleen, skeletal muscle and pancreas, P43 is abundantly produced in the liver and in other tissues such as the kidney, heart and brain which have levels of oxidative metabolism. Interestingly, a related protein of higher molecular weight is abundantly expressed in the lung and in amounts which were higher than those observed with other tissues. The human cDNA for P43 was isolated from a human liver cDNA library and mapped to chromosome 16 between p11.2 and 12 and also to a position near the centromere on the long arm of chromosome 17. The deduced amino acid sequence of P43 is remarkably similar to that of E. coli EF-Tu and the mitochondrial EF-Tu of S. cerevisiae with the structurally and functionally important amino acids of EF-Tu being completely conserved in P43. A comparison of the distribution of P43 and a mitochondrial protein Hsp 60 among different cellular fractions indicated a likely mitochondrial localisation for P43. Taken together these results suggest that P43 is a human mitochondrial elongation factor.

Amino Acid Sequence↗
PubMed1998

Sodium butyrate stimulates PKC activation and induces differential expression of certain PKC isoforms during erythroid differentiation.

Sodium butyrate (NaB) is an differentiation inducer currently under clinical investigation as a potential therapy for the treatment of sickle cell disease and prostate cancer. Though the biologic effects of this agent is well documented, its mechanism of action remains largely known. The mechanisms by which it transduces its signal to the nucleus is the subject of intense investigation in our laboratory. In this report, we demonstrate that NaB stimulates PKC activation by 3-fold and induces differential expression of several PKC isoforms. Notably, it upregulates PKC epsilon and downregulates PKC beta during erythroid differentiation. These findings suggest that certain PKC isoforms may play important roles in the signal transduction mechanisms of this agent leading to regulation of erythroid proliferation and differentiation.

Butyrates↗
PubMed2002

Identification of differentially expressed proteins between human esophageal immortalized and carcinomatous cell lines by two-dimensional electrophoresis and MALDI-TOF-mass spectrometry.

AIM: To identify the differentially expressed proteins between the human immortalized esophageal epithelial cell line (SHEE) and the malignant transformed esophageal carcinoma cell line (SHEEC), and to explore new ways for studying esophageal carcinoma associated genes. METHODS: SHEE and SHEEC cell lines were used to separate differentially expressed proteins by two-dimensional electrophoresis. The silver-stained 2-D gels was scanned with EDAS290 digital camera system and analyzed with the PDQuest 6.2 Software. Six spots in which the differentially expressed protein was more obvious were selected and analyzed with matrix-assisted laser desorption/ionization time of flying mass spectrometry (MALDI-TOF-MS). RESULTS: There were 107+/-4.58 and 115+/-9.91 protein spots observed in SHEE and SHEEC respectively, and the majority of these spots between the two cell lines matched each other (r=0.772), only a few were expressed differentially. After analyzed by MALDI-TOF-MS and database search for the six differentially expressed proteins, One new protein as well as other five sequence-known proteins including RNPEP-like protein, human rRNA gene upstream sequence binding transcription factor, uracil DNA glycosylase, Annexin A2 and p300/CBP-associated factor were preliminarily identified. CONCLUSION: These differentially expressed proteins might play an importance role during malignant transformation of SHEEC from SHEE. The identification of these proteins may serve as a new way for studying esophageal carcinoma associated genes.

Cell Line, Transformed↗
PubMed2002

Differential expression of retinoic acid-synthesizing (RALDH) enzymes during fetal development and organ differentiation in the mouse.

Three retinaldehyde dehydrogenases (RALDH1, RALDH2 and RALDH3), which catalyze the oxidation of retinaldehyde into retinoic acid, have been shown to be differentially expressed during early embryogenesis. Here, we report their differential expression patterns throughout later mouse organogenesis. Raldh1 is prominently expressed in developing lung (notably in bronchial and tracheal epithelia), and shows stage-specific expression in stomach and intestine epithelial and mesenchymal layers. Raldh3 expression is specific to the differentiating intestinal lamina propria. Raldh2 is expressed throughout the kidney nephrogenic zone, whereas Raldh1 and Raldh3 are mostly expressed in collecting duct epithelia. Raldh3 expression is more restricted than that of Raldh1 in the urogenital tract and sex gland epithelia, whereas Raldh2 expression is mesenchymal. Raldh1 is coexpressed with Raldh2 in the early heart epicardium, and is later specifically expressed in developing heart valves. All three genes exhibit distinct expression patterns in respiratory and olfactory epithelia and/or mesenchymes, and in developing teeth. Only Raldh1 expression is seen after birth in specific brain structures. These data indicate a requirement for regulated RA synthesis in various differentiating organs.

Abdomen↗
PubMed1997

The retinoblastoma gene family is differentially expressed during embryogenesis.

We report differential expression of the RB1 tumor suppressor gene and the homologous genes p107 and p130 during embryogenesis. Abundant RB1 transcripts were detected during neurogenesis, hematopoiesis, myogenesis, lens development and in the ganglion cell layer of the embryonic retina, prior to and during differentiation. The expression pattern of RB1 mirrored the defects in RB1 mutant mice (RB-/-). In the heart, lung, kidney and intestine, p107, but not RB1, was expressed. In the liver and the central nervous system p107 and RB1 were co-expressed, consistent with the accelerated cell death observed in RB-/-; p107-/- double knock-out mice. In the central nervous system, p107 expression was restricted to proliferating cells surrounding the ventricles, while RB1 was expressed in areas of both proliferating and differentiating cells. In contrast to RB1 and p107, expression of p130 was low throughout embryogenesis. In situ hybridization and Western blot analyses showed that the expression of p107 and p130 was not markedly altered in RB-/- embryos compared to control littermates. Our results suggest that members of RB1 gene family have distinct, but overlapping roles in embryogenesis, with p107 and RB1 possibly having redundant functions in the central nervous system and liver.

Animals↗
PubMed2003

[Identification of differentially expressed genes of acute hypoxia-treated HepG2 cells and hypoxia-acclimatized HepG2 cells].

To provide necessary information for further understanding of molecular mechanism of hypoxia acclimatization, the differentially expressed genes of HepG2 cells exposed to normoxia, acute hypoxia-treated cells which were exposed to 1% oxygen for 48 h, and hypoxia-acclimatized HepG2 cells which were cultured for 6 circles of alternate low oxygen (1% oxygen for 24 h) and normal oxygen (21% oxygen for 24 h), were identified respectively by combining the suppression subtractive hybridization (SSH) and cDNA microarray. Thirty-seven genes were expressed differentially in cells exposed to 1% oxygen for 48 h compared with those in cells exposed to normoxia. The expression of all these 37 genes was down-regulated, including the genes participating in cell cycle, cell response to stimulus, and cell signal transduction, and cell cytoskeleton formation, the genes associated with transcription and cell metabolism, 4 expressed sequence tags (ESTs), and 12 genes of which the functions are not known. There is a novel gene sequence, which has not been found in existing databases. There were only 6 genes differentially expressed in the hypoxia-acclimatized cells compared with cells exposed to normoxia, including two mitochondrion genes, metalloprotease-1 gene, ferritin gene, thymosin beta-4 and TPT1 genes. The expressions of mitochondrion ND4, ferritin, thymosin beta-4 and TPT1 were up-regulated, while the expressions of mitochondrion ND1 gene and metalloproease-1 gene were down-regulated. Cell tolerance to hypoxia increased after the cells were hypoxia-acclimatized. The different gene expression patterns of the acute hypoxia-treated cells and the hypoxia-acclimatized cells may be related to the increased tolerance of the cells to hypoxia.

Adaptation, Physiological↗
PubMed1999

Identification of true differentially expressed mRNAs in a pair of human bladder transitional cell carcinomas using an improved differential display procedure.

Differential display in combination with arbitrarily primed polymerase chain reaction (PCR) fingerprinting has become one of the most powerful techniques to identify and isolate mRNAs that are differentially expressed in pairs of biological samples. However, in many cases the cDNA band corresponding to the differentially amplified product contains several cDNA species that comigrate with the cDNA of interest due to the poor resolution of the fingerprinting gels, thus hampering further analysis and identification of the desirable cDNA. To improve the electrophoretic resolution of differentially amplified cDNAs, we have utilized Resolver Gold agarose gel electrophoresis (Ingenius) as an additional step to overcome downstream problems encountered during RNA fingerprinting experiments. To illustrate the power of the modified differential display procedure we present a detailed analysis of the cDNA products differentially displayed in tumor biopsies obtained from a noninvasive (grade II, Ta) and an invasive (grade III, T2-T4) human bladder transitional cell carcinoma (TCC). Several genes that were differentially expressed in this tumor pair were identified. These included: tropomyosin 4, the protein disulfide isomerase precursor (PDI), MRP14, signal transducer CD24, keratins 8 and 13, cytochrome oxidase subunit IV (COXIV), putative transcription factor HOX-1.3, as well as two novel genes of yet unknown function. All of the identified cDNAs were shown to be truly differentially expressed by Northern blotting, reverse transcriptase-PCR (RT-PCR), and two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) analysis of the corresponding lesions.

Bisbenzimidazole↗