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PubMed1997

A high throughput screening for rarely transcribed differentially expressed genes.

A novel method combining elements of suppression subtractive hybridization with high throughput differential screening permits the efficient and rapid cloning of rarely transcribed differentially expressed genes. The experimental strategy virtually excludes the possibility of isolating false positive clones. The potential of the method is demonstrated by the isolation of 625 differentially expressed cDNAs from the metastatic adenocarcinoma cell line Bsp73-ASML when subtracted from its non-metastatic counterpart Bsp73-1AS. Northern analysis of 72 randomly selected clones demonstrated that 68 were differentially expressed with respect to Bsp73-ASML, indicating a true positive rate of 94%. Additionally, a large proportion of these clones represented rare transcripts as determined by the exposure time required to detect a signal. Sequence data indicated that of the 625 clones obtained, 92 clones scored perfect or near perfect matches with already known genes. Two hundred and eighty one clones scored between 60 and 95% homology to known human and mouse genes, whereas 252 clones scored no match with any sequences in the public databases. The method we describe is ideally suited whenever subtle changes in gene expression profiles need to be determined.

Adenocarcinoma↗

PubMed2005

Differential expression and cytoplasm/membrane distribution of endoglin (CD105) in human tumour cell lines: Implications in the modulation of cell proliferation.

Endoglin (CD105, an accessory component of the TGF-beta receptor complex) expression and distribution on different human tumour cells and its role in cellular proliferation were evaluated. We examined: 1) sixteen human carcinoma cell lines, 2) eight human sarcoma cell lines, 3) five miscellaneous tumour cell lines. HECV (endothelial cells) were employed as a positive control for endoglin expression. Normal Human Dermal Fibroblasts (NHDF) and 293 cells (epithelial kidney cells) were used as normal controls for connective and epithelial tissues, respectively. The results showed that CD105 was poorly expressed in the majority of human carcinoma cells (10/16), whereas it was highly expressed in most human sarcoma cells (7/8), and differently expressed by miscellaneous tumour cell lines. These data reflect endoglin expression by the normal counterparts of tumour cell lines, i.e. NHDF and 293 cells. However, CD105 levels in sarcoma cell lines, even though consistently lower than in NHDF, were significantly higher than those observed in carcinoma cells. Interestingly, CD105 presented a strong expression in the cytoplasm of MDA-MB-453 (breast carcinoma), NPA (papillary thyroid carcinoma), COLO-853 (melanoma) and SaOS-2 (osteosarcoma), but was weakly expressed on their cell membrane. This differential expression in the cytoplasm and on the membrane of some tumour cells, suggests a complex mechanism of translocation for this protein. The analysis of clonal growth in soft agar of some cell lines, characterized by high CD105 expression, showed an increased colony formation potential that was antagonized by the addition of anti-CD105 blocking mAb. The results indicated that endoglin is differentially expressed in human carcinoma and sarcoma cells and its overexpression modulates the proliferative rate of human solid tumour cells. Moreover, these data suggest that CD105 is involved in the regulation of TGF-beta effects in human solid malignancies, and therefore it could play an important role in tumour diagnosis and treatment.

Antigens, CD↗

PubMed2005

[CDNA microarray on differentially expressed genes of adipose tissue in two breeds chicken].

cDNA microarray containing 9024 cDNAs was used to construct gene expression profile in order to screen differentially expressed genes of adipose tissue between broiler and Bai' er and investigate the molecular mechanism related with body fatness traits between the two breeds. Sixty seven differentially expressed genes, being involved in fat metabolism, energy metabolism, cytoskeleton, transcription and splicing factor, protein synthesis and degradation, were screened out. Furthermore, some genes that had no annotation in GenBank were screened out, they were presumed to be unknown new genes. The roles that they may play in chicken fat metabolism need clarify later.

Adipose Tissue↗

PubMed2001

Human EGF receptor (HER) family and heregulin members are differentially expressed in epidermal keratinocytes and modulate differentiation.

Human EGF receptor (HER), also designated HER1, is an activatable tyrosine kinase receptor. HER1 activation regulates cell growth and differentiation in epidermal keratinocytes. Expression of other HER family members was investigated in human keratinocytes cultured under autocrine conditions. HER2 and HER3 are expressed and upregulated by confluence, concurrent with induction of epidermal differentiation. HER4 is not expressed by keratinocytes. Maximum expression of the cognate ligand, heregulin, is observed in subconfluent keratinocytes, and the expression of both heregulin alpha and beta isoforms is downregulated with confluence. Recombinant heregulin isoforms have no effect on colony formation and keratinocyte proliferation, but heregulin beta activates tyrosine phosphorylation of HER2 and HER3, with no effect on HER1, in confluent differentiating keratinocyte cultures. Also, heregulin beta increases HER2/HER3 heterodimerization under those conditions. Treatment of confluent cultures by heregulin beta correlates with cell signaling and inhibition of epidermal differentiation. Together, HER2, HER3, and heregulin constitute a potential autocrine-paracrine system involved in epidermal homeostasis and repair, as well as in hyperproliferative pathologies.

Autocrine Communication↗

PubMed2005

Genome-wide analysis of differentially expressed genes from Penicillium chrysogenum grown with a repressing or a non-repressing carbon source.

Penicillium chrysogenum is an economically important ascomycete used as industrial producer of penicillin. However, with the exception of penicillin biosynthesis genes, little attention has been paid to the genetics of other aspects of the metabolism of this fungus. In this article we describe the first attempt of systematic analysis of expressed genes in P. chrysogenum, using a suppression subtractive hybridization approach to clone and identify sequences of genes differentially expressed in media with glucose or lactose as carbon source (penicillin-repressing or non-repressing conditions). A total of 167 clones were analysed, 95 from the glucose condition and 72 from the lactose condition. Genes differentially expressed in the glucose condition encode mainly proteins involved in the mitochondrial electron transport chain and primary metabolism. Genes expressed differentially in lactose-containing medium include genes for secondary metabolism (pcbC, isopenicillin N synthase), different hydrolases and a gene encoding a putative hexose transporter or sensor. The results provided information on how the metabolism of this fungus adapts to different carbon sources. The expression patterns of some of the genes support the hypothesis that glucose induces higher rates of respiration in P. chrysogenum while repressing secondary metabolism.

Amino Acid Sequence↗

PubMed1995

Identification and partial sequence of a cDNA that is differentially expressed in human brain tumors.

Differential display technique was used to identify mRNAs that are differentially expressed in malignant versus benign brain tumors. Using this method, a novel 1.4-kb long cDNA (MM1) clone was isolated and sequenced. The nucleotide and the translated amino acid sequence of MM1 cDNA clone did not show significant homology to any known sequence in the Genebank. The expression of MM1 appears to be almost eightfold higher in glioblastomas compared to low grade astrocytomas and slightly higher in malignant meningiomas than in benign meningiomas. The size of mRNA from northern analysis appears to be 7 kb, which is much higher than the size of the isolated MM1 cDNA clone. Expression of MM1 was also seen in various cell lines derived from human tumors including glioblastomas. Whereas low level expression was seen in kidney, esophagus, liver, lymph node, ovary and testis, none of the other tissues from a total of 18 different human organs showed any MM1 expression.

Amino Acid Sequence↗

PubMed2003

[Differentially expressed genes in hepatocellular carcinoma of tree shrew induced by different factors].

BACKGROUND & OBJECTIVE: Previous studies on differentially expressed genes in hepatocellular carcinoma (HCC) used to perform with para-cancerous tissues as normal control. However, the para-cancerous tissue of HCC is actually abnormal because they frequently contain hepatitis, cirrhosis, hyperplastic nodules or foci, etc. In order to explore the molecular mechanism and the responsible genes for hepatocarcinogenesis, through applying the HCC model of tree shrew (Tupaia belangeri chinensis), this study was designed to compare gene expression levels between HCC induced by different factors and their corresponding biopsies taken before HCC formation. METHODS: Tree shrews were divided into two groups. Group AFB(1) was fed with aflatoxin B1 (AFB(1)). Group AFB(1)+HBV was infected firstly with human hepatitis B virus (HBV) and then fed with AFB(1) as group AFB(1). Serum tests for HBV markers and liver biopsies were performed periodically during the experiment. After appearance of HCC, 2 HCC samples from each group and their corresponding 30th-week biopsies were tested respectively by cDNA microarray assay. The gene expression levels were compared between each HCC and the corresponding biopsies, and the differentially expressed genes from the two groups of HCC induced by different factors were analyzed. RESULTS: The incidence rates of HCC in group AFB(1) and group AFB(1)+HBV were 73.3% and 77.8%, respectively. A considerable number of genes in both groups showed changes in their expression levels, which were mainly up-regulated in group AFB(1) but down-regulated in group AFB(1)+HBV. On the other hand, among the 588 checked genes (16 functional classifications) that were known related to human cancer, 11 genes were similarly expressed in all of the 4 HCC from the two animal groups. Most of these 11 genes belonged functionally to 3 types, namely "apoptosis-associated protein","DNA synthesis,repair and recombination proteins", and "growth factors, cytokines and chemokines". CONCLUSION: (1)HBV can affect AFB(1)-induced gene expression in certain extent. (2)The gene expression profiles of HCC induced by different factors are different. (3)The common differentially expressed genes in these two HCC groups are worthwhile for further study as the possibly responsible genes for hepatocarcinogenesis.

Aflatoxin B1↗

PubMed1998

Differential screening and suppression subtractive hybridization identified genes differentially expressed in an estrogen receptor-positive breast carcinoma cell line.

Differences in gene expression are likely to explain the phenotypic differences between hormone-responsive and hormone-unresponsive breast cancer. We have identified differentially expressed cDNAs in the estrogen receptor (ER)-positive MCF7 breast carcinoma cell line compared with the ER-negative MDA-MB-231 breast carcinoma cell line. Differential screening isolated four differentially expressed genes: cytokeratin 8, cytokeratin 18, Hsp27 and GPCR -Br. To identify differentially expressed genes of lower abundance, suppression subtractive hybridization was utilized and 29 differentially expressed clones were isolated. Sequence analysis revealed that 11 clones were from previously described genes: HEK8, neuropeptide Y receptor Y1, p21 WAF-1, p55 PIK, cytokeratin 18 (cloned twice), fructose-1,6-biphosphatase, cytokeratin 8, TGFbeta1 binding protein, elongation factor 1alpha2 and pS2. The remaining 18 clones did not match sequences in the GenBank/EMBL database, indicating that they may be novel genes. Expression of pS2, neuropeptide Y receptor Y1 and three novel clones was induced by estradiol, indicating estrogen-responsiveness. The expression pattern of one novel gene, DEME -6, correlated with expression of ER and ERF -1/ AP -2gamma in a panel of breast carcinoma cell lines. A 2.6 kb cDNA of DEME -6 was sequenced and contains an open reading frame of 574 amino acids that demonstrates 62.4% similarity with a gene from Caenorhabditis elegans chromosome III. Expression of DEME -6 was also detected in primary breast carcinomas but not in normal breast tissue, as determined by RT-PCR. These findings support the hypothesis that a set of genes coordinately regulated with ER , but not necessarily estradiol-responsive, are characteristic of the hormone-responsive breast cancer phenotype.

Amino Acid Sequence↗

PubMed2004

Identification of genes differentially expressed in rat alveolar type I cells.

Although approximately 98% of the internal surface area of the lung is lined by alveolar type I cells, little is known about the functions of this cell type. Using freshly isolated rat type I and type II cells, we created a subtraction library by suppression subtractive hybridization to identify genes differentially expressed by type I cells. We identified twelve genes of known function that are differentially expressed by type I cells. Differential expression of all 12 genes was confirmed by Northern blotting; we confirmed differential expression by immunocytochemistry for 3 genes for which suitable antibodies were available. Most of the genes code for proteins that are multifunctional. From the known functions of these genes, we infer that type I cells may play a role in the maintenance of normal alveolar homeostasis and protection from injury, lung development and remodeling, host defense, tumor/growth suppression, and surfactant metabolism, among other functions.

Animals↗

PubMed2005

Screening and identification of differentially expressed transcripts in circulating cells of prostate cancer patients using suppression subtractive hybridization.

BACKGROUND: Tumor metastasis and changes in host immunosurveillance are important components in cancer development. Tumor cell invasion into the bloodstream is an essential step for systemic metastasis. Currently, the detection of tumor cells in the circulation is mainly dependent upon the utilization of known epithelial cell markers. However, expression of these molecules is not limited to cancer patients; healthy people also have a small number of epithelial cells in their circulation. Utilizing these markers to detect circulating tumor cells (CTCs) cannot adequately explain the mechanisms of tumor cell survival or their development of metastatic potential in peripheral blood. The immune system can also evolve along with the cancer, actually promoting or selecting the outgrowth of tumor variants. Unfortunately, both metastasis and immunosurveillance remain mysterious and are debatable because we have yet to define the molecules that participate in these processes. We are interested in identifying the existence of expressed genes, or mRNA species, that are specifically associated with circulating cells of cancer-bearing patients using prostate cancer (PCa) as a model. RESULTS: We established two comprehensive subtracted cDNA libraries using a molecular technique called suppression subtractive hybridization. This technique selectively amplifies transcripts that are specifically expressed in circulating cells of either PCa patients or healthy men. Following sequencing reaction, we showed that 17 out of 23 (73.9%) sequenced clones did not match any mRNAs in the GenBank database. This result suggests that genes associated with alterations in circulating cells of cancer-bearing patients are largely unknown. Semi-quantitative RT-PCR confirmed that two genes are up-regulated in circulating cells of PCa patients, whereas another two genes are down-regulated in the same patients. CONCLUSION: The comprehensive gene expression analysis is capable of identifying differentially expressed genes in circulating cells of healthy men and PCa patients. We did not attempt to enrich specific cell types in this study because phenotypes of CTCs and subsets of leukocytes participating in immunosurveillance remain largely unknown. Continuous studies of these differentially expressed genes will eventually lead us to understand the mechanisms involved in tumor metastasis and immune modulation during cancer development.

Biomarkers, Tumor↗

PubMed2005

The analysis of differentially expressed novel transcripts in diapausing and diapause-activated eggs of Bombyx mori.

As an initial step to define the molecular mechanism of initiation and termination of diapause during the embryogenesis of silkworms, Bombyx mori, mRNA transcripts from maintained and activated diapause eggs were compared with differential expression using cDNA array. Twenty-four individual cDNA transcripts were expressed differentially in a total of 1,468 different cDNAs. Among those clones, mRNA transcript from cytochrome oxidase subunit I (COI), which was detected to be 2-kb transcripts, gradually increased in diapause-activated eggs during early embryogenesis. Further analysis revealed that mRNA transcripts from silkworm COI were highly expressed in testis, fat body, and midgut during the larval stage. These results may indicate that the expression of silkworm COI mRNA is regulated developmentally as well as tissue-specifically.

Animals↗

PubMed2004

Accentuation of differentially expressed proteins using phage technology.

Protein profiling is frequently used to elucidate disease-specific or differentially expressed proteins. While recent developments have resulted in improved differential profiling, alternative expression platforms that complement existing techniques are continually being explored. We developed a novel method utilizing the amplification and selection capabilities of random peptide-expressing M13 bacteriophage to accentuate differentially expressed proteins in biologic specimens. While the current study used this method to demonstrate differentially expressed proteins in lung cancer tissue in comparison to normal lung tissue, this approach is applicable to a wide range of sample types.

Bacteriophage M13↗

PubMed2006

Multifunctional regulators of cell growth are differentially expressed in anergic murine B cells.

Defective anergy is a major cause of failed tolerance and is amenable to therapeutic manipulation. To better define the molecular basis of anergy in B cells tolerized by matrix self-antigen, we used complementary approaches of representational difference analysis (RDA) and microarray to identify genes differentially transcribed in anergic as compared to non-tolerant B cells isolated from a well-characterized murine autoantibody transgenic model. Forty RDA clones representing 16 genes were isolated from receptor-stimulated B cells and independently confirmed as differentially expressed in tolerant cells using custom microarray, dot blotting and/or quantitative PCR. Differential expression was conserved in tolerant cells from two different transgenic founder lineages and from two genetically disparate backgrounds. Prominent among recovered gene fragments were genes encoding multifunctional proteins not previously implicated in B cell biology, but with roles in biologic processes fundamental to the tolerance phenotype, including cell growth, proliferation and differentiation. RDA also identified a novel transcript not previously reported in nucleic acid databases. To further explore dependence on receptor stimulation and to identify additional genes, commercial oligonucleotide arrays were probed with labeled B cell transcripts and analyzed for genes differentially expressed in resting as well as stimulated cells and in both B6 and MRL mouse strains. Arrays identified differential expression of a subset of RDA genes as well as 46 additional genes, including subsets engaged in signal transduction, transcriptional regulation, cell growth and apoptosis. Immunoblotting confirmed differential protein expression for galectin-3 and galectin-1, two interactive members of the galectin family, suggesting a novel role for galectins as regulators of immune tolerance.

Animals↗

PubMed1998

Differentially expressed genes in C6.9 glioma cells during vitamin D-induced cell death program.

C6.9 rat glioma cells undergo a cell death program when exposed to 1, 25-dihydroxyvitamin D3 (1,25-D3). As a global analytical approach, we have investigated gene expression in C6.9 engaged in this cell death program using differential screening of a rat brain cDNA library with probes derived from control and 1,25-D3-treated cells. Using this methodology we report the isolation of 61 differentially expressed cDNAs. Forty-seven cDNAs correspond to genes already characterized in rat cells or tissues. Seven cDNAs are homologous to yeast, mouse or human genes and seven are not related to known genes. Some of the characterized genes have been reported to be differentially expressed following induction of programmed cell death. These include PMP22/gas3, MGP and beta-tubulin. For the first time, we also show a cell death program induced up-regulation of the c-myc associated primary response gene CRP, and of the proteasome RN3 subunit and TCTP/mortalin genes. Another interesting feature of this 1,25-D3 induced-cell death program is the down-regulated expression of transcripts for the microtubule motor dynein heavy chain/MAP 1C and of the calcium-binding S100beta protein. Finally 15 upregulated cDNAs encode ribosomal proteins suggesting a possible involvement of the translational apparatus in this cell program. Alternatively, these ribosomal protein genes could be up-regulated in response to altered rates of cellular metabolism, as has been demonstrated for most of the other isolated genes which encode proteins involved in metabolic pathways. Thus, this study presents to our knowledge the first characterization of genes which are differentially expressed during a cell death program induced by 1, 25-D3. Therefore, this data provides new information on the fundamental mechanisms which participate in the antineoplastic effects of 1,25-D3 and on the machinery of a cell death program in a glioma cell line.

Animals↗

PubMed2006

[Differentially expressed genes between a fertile patient and an infertile patient in a large Chinese androgen insensitivity syndrome pedigree].

OBJECTIVE: To screen and identify differentially expressed genes between a fertile patient and another infertile patient who belonged to a large Chinese pedigree affected with androgen insensitivity syndrome (AIS). METHODS: We constructed the forward and reversed subtracted libraries using genital skin fibroblasts (GSF), which were obtained from the fertile patient MJ and infertile patient ZGJ, as tester respectively. Candidate clones were screened with colony in situ hybridization, dot blot, and Southern blot analysis step by step and conformed with Northern blot analysis. The potential positive clones were sequenced and the homology of the sequences was analyzed. RESULTS: The forward and reversed subtracted libraries containing differentially expressed pattern of two GSF cell lines were constructed. Two positive clones identified by Northern blot were obtained in the reversed subtracted library. Eleven candidate clones from the two libraries that failed to hybridize with both RNA populations were obtained simultaneously, which might represent differentially expressed low abundance transcripts. Sequencing results and homology analysis demonstrated that the two positive clones were significantly homologous with the genes of autotaxin-t and calcium binding protein calcyclin (S100A6), respectively. CONCLUSIONS: Two positive clones and eleven clones showing no hybridization signals may represent differentially expressed genes between the two GSFs. This finding may be useful to elucidate the molecular mechanisms leading to phenotypic variation and preserved fertility of the AIS pedigree.

Androgen-Insensitivity Syndrome↗

PubMed2003

Differential expression of L- and S-MAG upon cAMP stimulated differentiation in oligodendroglial cells.

Myelin-associated glycoprotein (MAG), an immunoglobulin-like cell signaling protein involved in axon-glial interactions, displays two intracellular C-termini as a result of alternative mRNA splicing. During brain development, the two MAG mRNAs that encode L-MAG and S-MAG differ in their relative abundance. We have investigated the differential expression of L- and S-MAG upon cAMP treatment in the oligodendroglial cell line Oli-neu, a cell line able to differentiate in vitro. We have engineered GFP and VSVG fusions by small insertions into the alternatively spliced exons of the cloned MAG gene and reintroduced them into Oli-neu cells. The individually tagged MAG isoforms were expressed under the control of the MAG promoter and regulatory region. In this system, L-MAG was the predominant isoform before the stimulation of cells with cAMP, whereas upon cAMP treatment the S-MAG isoform was predominantly expressed in cells with a high degree of morphological differentiation. We suggest that the regulation of the MAG alternative splicing and the morphological differentiation in oligodendrocytes are controlled both by the same cAMP-responsive differentiation step.

Animals↗

PubMed1992

Differential expression of a novel murine non-receptor protein tyrosine phosphatase during differentiation of P19 embryonal carcinoma cells.

Protein phosphorylation on tyrosine residues is one of the major mechanisms of cell signal transduction and is regulated by protein tyrosine kinases and protein tyrosine phosphatases. Here we report the molecular cloning of an additional member of the protein tyrosine phosphatase-family from differentiated murine P19 embryonal carcinoma cells. This non-receptor protein tyrosine phosphatase, P19-PTP, does not contain regulatory sequences, homologous to the ones found in other non-receptor PTPases. P19-PTP is differentially expressed during in vitro differentiation of P19 EC cells, in that P19-PTP mRNA could only be detected in embryoid bodies, derived from P19 cells.

Amino Acid Sequence↗

PubMed2000

Distribution of uridine diphosphate-glucuronosyltransferase (UGT) expression and activity in cynomolgus monkey tissues: evidence for differential expression of steroid-conjugating UGT enzymes in steroid target tissues.

Based on the similarity of pathways and enzymes involved in steroid metabolism, simians represent a relevant animal model to study steroid elimination by glucuronidation. In this study the tissue distribution of UDP-glucuronosyltransferase (UGT) transcripts, proteins, and enzymatic activities were examined in 24 different cynomolgus monkey tissues. RT-PCR and Western blot analysis on total RNA and microsomal proteins demonstrated the presence of UGT1A and UGT2B transcripts and proteins in a wide range of tissues including steroid target tissues. Glucuronidation activity on eugenol, 5alpha-androstane-3alpha,17beta-diol, androsterone, and 4-hydroxyestradiol was measured using tissue homogenates and radiolabeled [14C]UDP-glucuronic acid. All tissues contained conjugation activity on these substrates, but glucuronidation rates were significantly lower in steroid target tissues than in liver, kidney, or gut. However, the ratio of steroid glucuronidation vs. eugenol glucuronidation was higher in steroid target tissues, suggesting a differential expression of steroid-conjugating enzymes in these tissues. Taken together, these results clearly demonstrate the presence of steroid glucuronidation enzymes in extrahepatic steroid target tissues and support the hypothesis that steroid glucuronidation is an important intracrine pathway involved in termination of steroid signaling.

Animals↗