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PubMed2004

Host cell gene expression during human immunodeficiency virus type 1 latency and reactivation and effects of targeting genes that are differentially expressed in viral latency.

The existence of reservoirs of cells latently infected with human immunodeficiency virus (HIV) is a major obstacle to the elimination of HIV infection. We studied the changes in cellular gene expression that accompany the reactivation and completion of the lytic viral cycle in cell lines chronically infected with HIV-1. We found that several genes exhibited altered expression in the chronically infected cells compared to the uninfected parental cells prior to induction into lytic replication. A number of gene classes showed increased expression in the chronically infected cells, notably including genes encoding proteasomes, histone deacetylases, and many transcription factors. Following induction of the lytic replication cycle, we observed ordered, time-dependent changes in the cellular gene expression pattern. Approximately 1,740 genes, many of which fall into 385 known pathways, were differentially expressed (P < 0.001), indicating that completion of the HIV replication cycle is associated with distinct, temporally ordered changes in host cell gene expression. Maximum changes were observed in the early and intermediate phases of the lytic replication cycle. Since the changes in gene expression in chronically infected cells suggested that cells latently infected with HIV have a different gene expression profile than corresponding uninfected cells, we studied the expression profiles of three different chronically infected cell lines to determine whether they showed similar changes in common cellular genes and pathways. Thirty-two genes showed significant differential expression in all cell lines studied compared to their uninfected parental cell lines. Notable among them were cdc42 and lyn, which were downregulated and are required for HIV Nef binding and viral replication. Other genes previously unrelated to HIV latency or pathogenesis were also differentially expressed. To determine the effects of targeting products of the genes that were differentially expressed in latently infected cells, we treated the latently infected cells with a proteasome inhibitor, clastolactacystin-beta-lactone (CLBL), and an Egr1 activator, resveratrol. We found that treatment with CLBL and resveratrol stimulated lytic viral replication, suggesting that treatment of cells with agents that target cellular genes differentially expressed in latently infected cells can stimulate lytic replication. These findings may offer new insights into the interaction of the latently infected host cell and HIV and suggest therapeutic approaches for inhibiting HIV infection and for manipulating cells latently infected with HIV so as to trigger lytic replication.

Cell Line↗
PubMed1992

Differential expression of PKC isoforms and PC12 cell differentiation.

Recent reports indicate that the protein kinase inhibitor H7 is capable of inducing both morphological and functional differentiation of a number of neural cell types. This investigation demonstrates that H7 potentiates the neurogenic properties of nerve growth factor (NGF) in PC12 cells with a concomitant change in the accumulation of the beta II-protein kinase C (beta IIPKC) isoform protein without changes in either alpha or gamma. However, NGF alone stimulates a coordinate increase in all three isoforms. The assay of acetylcholine esterase as a functional marker of neuronal differentiation demonstrates that H7 alone is not capable of stimulating morphological or functional differentiation in PC12 cells. H7 synergizes with NGF through a PKC-dependent pathway and by differential expression of PKC subtypes. The expression of the PKC transcripts for alpha, beta II, and gamma all undergo simultaneous yet differential changes in their patterns of expression during treatment with H7 and/or NGF. These data suggest that isoform switching is regulated primarily at the protein level. Last, these findings suggest that expression of PKC isoforms is tightly coupled with neuronal differentiation and may play a role in the maintenance of the differentiated state.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed2000

Differentially expressed genes during in vitro differentiation of murine embryonic stem cells transduced with a human erythropoietin receptor cDNA.

Our previous study demonstrated that transduction of murine embryonic stem (ES) cells with a human erythropoietin (Epo) receptor (R) cDNA resulted in enhanced erythropoiesis in developing embryonic bodies (EBs). To address possible mechanisms of gene regulation, we compared gene expression between hEpoR cDNA-transduced ES (ES-hEpoR) cells and parental ES cells during in vitro differentiation induced by withdrawal of leukemia inhibitory factor (LIF) and cultured in the absence of Epo using differential display reverse transcriptase-polymerase chain reaction (DDRT-PCR). A total of 48 differentially expressed cDNA fragments were found; 12 were sequenced and five were confirmed by Northern blot analysis to be up- or down-regulated in ES-hEpoR cells during differentiation compared to parental ES cells. In a GenBank search of the five putatively regulated cDNA fragments, two fragments shared high sequence homology to two known genes: the Surf-6 gene and the gene for calcyclin binding protein. Northern blot analysis demonstrated that 2.5-kb and 0.3-kb transcripts of the Surf-6 gene were expressed in undifferentiated ES-hEpoR and parental ES cells at a low level, but this expression was enhanced from day 2 to 14 of differentiation after withdrawal of LIF and culture in the presence of Epo. Furthermore, the enhanced expression of these two transcripts was also noticed in EML-C1 cells, a murine multipotential hematopoietic cell line that has erythroid differentiation potential in response to Epo. In summary, our results demonstrate that Surf-6 gene expression is regulated during differentiation of hematopoietic stem/progenitor cells in response to Epo, suggesting a possible role for Surf-6 gene in erythropoiesis.

Animals↗
PubMed1995

Identification of differentially expressed genes by restriction endonuclease-based gene expression fingerprinting.

A novel method for identification of differentially expressed genes has been developed. It is based on the consecutive restriction digestions of 3' terminal cDNA fragments to produce a fingerprint of gene expression. cDNA molecules are synthesized using a biotinylated oligo(dT) primer, digested with a frequently cutting restriction endonuclease and the 3'-terminal restriction fragments are isolated using streptavidin microbeads. After amplification by PCR, cDNA fragments are immobilized again on streptavidin beads, radiolabeled and treated sequentially with a set of restriction endonucleases. The products of individual enzymatic reactions from two or more different RNA populations are resolved by polyacrylamide gel electrophoresis and compared to reveal differentially expressed genes. This strategy enabled us to identify and clone the fragments of five genes expressed differentially in murine thymus and spleen. One of the genes was found to encode terminal deoxynucleotidyl transferase; others are apparently previously unknown genes.

Animals↗
PubMed2000

Gene expression profile and identification of differentially expressed transcripts during human intrathymic T-cell development by cDNA sequencing analysis.

The development of immature thymocytes to mature T-lymphocytes is a central process for establishing a functional immune system. The gene regulatory events involved in this process are of outstanding interest in understanding the generation of the T-cell repertoire as well as the differentiation of lineage-specific cells, such as CD4(+) helper T-cells or CD8(+) cytotoxic T-lymphocytes. While some essential genes involved in lineage decision and thymocyte differentiation have been already identified, the exact regulatory mechanisms and differential gene expressions are still unknown. The present study was performed to analyze the gene expression profile during T-cell development, in particular, during the differentiation of immature thymocytes into CD4(+) mature T-cells by analyses of expressed sequence tags (ESTs), and to elucidate novel human genes involved in this process. Based on distinct developmental stages, three PCR-based cDNA libraries from immature CD3(-),4(-),8(-) triple-negative, CD4(+),8(+) double-positive, and mature CD4(+),8(-) single-positive thymocytes were constructed. A total of 1477 randomly selected clones were analyzed by automated single-pass sequencing, and the assembly of ESTs resulted in 1027 different species of contig sequences. Among them, 392 contig sequences were matched to known genes, and several novel transcripts were discovered. The matched clones were classified into seven categories according to their functional aspects, and the gene expression profiles of the three thymocyte subsets were compared. The information obtained in current study will serve as a valuable resource for elucidating the molecular mechanism of intrathymic T-cell development.

Cell Differentiation↗
PubMed1986

Differential expression of T cell differentiation antigens and major histocompatibility antigens on activated T cells during the cell cycle.

In this report we have analyzed cell cycle-related fluctuations of both quantity and density of the T cell differentiation antigens, CD3 (T3), CD4 (T4) and CD8 (T8), as well as the major histocompatibility complex (MHC) antigens on the cell surface of activated T cells. Phytohemagglutinin-activated T cells cultured for 3 days with or without conditioned medium or for 10 days with conditioned medium and mixed lymphocyte culture-derived T cell clones were used for the analysis. Correlated measurements of the surface antigen quantity (immunofluorescence), DNA content (dye Hoechst 33342), and cell size (light scatter), not influenced by synchrony induction methods and cell fixation, were performed by dual-beam flow cytometry. Our results demonstrate that the T cell differentiation antigens, CD3, CD4 and CD8, and class I MHC antigens are increased in density in the G1 phase for all activated T cells tested. In contrast, class II MHC antigens are increased in density in the G2 phase of activated T cells maintained with conditioned medium. Since it is known that the T cell differentiation antigens and class I MHC antigens on activated T cells are necessary for proliferation of T cells, our study suggests that this effect is more significant in the G1 phase. The cell cycle changes in expression of class I and class II MHC antigens, but not of the T cell differentiation antigens, appear to be mediated by soluble factors, probably including interferon-gamma, which could produce a differential increase of class I and class II MHC antigens on G2 phase cells.

Antigens, Differentiation, T-Lymphocyte↗
PubMed2004

AF1q, a differentially expressed gene during neuronal differentiation, transforms HEK cells into neuron-like cells.

Three cell groups, neural stem/progenitor cells (NS/PCs) dissociated from the embryonic day 11 (E11) rodent cerebral cortex, expanded NS/PC cultures, and cultured neurons from E15, were used to conduct a genomic study with differential display (DD). The mouse Af1q, homologue of human AF1q, was found to be significantly up-regulated during the neuronal production from NS/PCs. The ectopic expression of human AF1q triggered the expression of the neuronal marker TuJ1 in non-neuronal human embryonic kidney (HEK) cells.

Animals↗
PubMed2003

[Screening and identification for cDNA of differentially expressed genes in human primary hepatocellular carcinoma].

OBJECTIVE: Screening and identification of differentially expressed genes in human primary hepatocellular carcinoma(HCC). METHODS: The differentially expressed genes subtracted cDNA library of HCC constructed by suppression subtractive hybridization(SSH) technique was screened by colony in situ hybridization, then the positive clones were further screened with PCR amplification. The positive clones were sequenced and analyzed for homology in the Genbank databases with Basic Local Alignment Search Tool BLAST . The novel cDNA sequences were analyzed by Northern blot analysis. RESULTS: Thirteen positive clones were obtained, and 11 cDNA sequences were identified. Sequences of 11 cDNA showed that 6 cDNA were homologous with the genes published in Genbank and 5 cDNA were unknown genes. Northern blot indicated that 3 novel cDNA(>300 bp) were only expressed in HCC. CONCLUSION: The subtracted cDNA library constructed by SSH technique contains differentially expressed genes of HCC. Three novel cDNA sequences might be differentially expressed genes of HCC. Further screening the library and gaining the whole gene sequence may lay a foundation for identifying differentially expressed genes in HCC.

Base Sequence↗
PubMed1998

Profiling of differentially expressed genes in human primary cervical cancer by complementary DNA expression array.

The profiling of differentially expressed genes from primary tumor samples using cDNA expression array can reveal new tumor markers as well as target genes for therapeutic intervention. Using cDNA expression array technology, we produced an expression profile of genes that are associated with human cervical cancer. Hybridization of the cDNA blotting membrane (588 genes on a single membrane) was performed with 32P-labeled cDNA probes synthesized from RNA isolated from either normal cervix or cervical cancer. Parallel analyses of the hybridized signals enabled us to profile genes that were differentially expressed in cervical cancer. In each experiment, the extent of hybridization of each gene was evaluated by comparison with the most abundant mRNAs in the human cervix. These include myc proto-oncogene, 40S ribosomal protein S19, heat shock proteins, leukosialin S (CD43), integrin alphaL (CD11A), calgranulin (A), and CDK4 inhibitor (p16ink4). No detectable changes were observed in the expression levels of these genes. Several mRNAs, such as those encoding guanine nucleotide-binding protein Gs (alpha subunit), leukocyte adhesion protein (LFA1-beta), nuclear factor NF45, homeobox protein Hox-A1, and beta-catenin were detected in increased levels in cervical cancer. Genes that showed decreased expression in cervical cancer tissue were a group of apoptosis-related proteins, cell adhesion molecules, nuclear transcription factors, and a homeobox protein (Hox7). For example, the expression levels of Smad1 and Hox7 were consistently decreased in all tumor tissues tested. Northern analysis of Smad1 and Hox7 RNA in primary cervical tumor tissues and cervical carcinoma cell lines indicated that, in general, the mRNA levels of these genes were decreased in human cervical cancer. The precise relationship between the altered expression of these genes and cervical tumorigenesis is a matter of further investigation.

DNA, Complementary↗
PubMed2005

Biological validation of differentially expressed genes in chronic lymphocytic leukemia identified by applying multiple statistical methods to oligonucleotide microarrays.

Oligonucleotide microarrays are a powerful tool for profiling the expression levels of thousands of genes. Different statistical methods for identifying differentially expressed genes can yield different results. To our knowledge, no experimental test has been performed to decide which method best identifies genes that are truly differentially expressed. We applied three statistical methods (dChip, t-test on log-transformed data, and Wilcoxon test) to identify differentially expressed genes in previously untreated patients with chronic lymphocytic leukemia (CLL). We used a training set of Affymetrix Hu133A microarray data from 11 patients with unmutated immunoglobulin (Ig) heavy chain variable region (VH) genes and 8 patients with mutated Ig VH genes. Differential expression was validated using semiquantitative real-time polymerase chain reaction assays and by validating models to predict the somatic mutation status of an independent test set of nine CLL samples. The methods identified 144 genes that were differentially expressed between cases of CLL with unmutated compared with mutated Ig VH genes. Eighty genes were identified by Wilcoxon test, 60 by t-test, and 65 by dChip, but only 11 were identified by all three methods. Greater agreement was found between the t-test and the Wilcoxon test. Differential expression was validated by semiquantitative real-time polymerase chain reaction assays for 83% of individual genes, regardless of the statistical method. However, the Wilcoxon test gave the most accurate predictions on new samples, and dChip, the least accurate. We found that all three methods were equally good for finding differentially expressed genes, but they found different genes. The genes selected by the nonparametric Wilcoxon test are the most robust for predicting the status of new cases. A comprehensive list of all differentially expressed genes can only be obtained by combining the results of multiple statistical tests.

Algorithms↗
PubMed2002

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

UNLABELLED: In searching of differentially expressed genes in human uterine leiomyomas, differential display was used with twelve pairs of primers to compare human uterine leiomyomas with matched myometrium. False positives were eliminated by reverse Northern analysis. Positives were confirmed by Northern blot analysis. RESULTS: Four of 69 cDNA fragments (3 up-regulated named L1, L2 and L3 and 1 down-regulated named M1 in leiomyoma) were confirmed by Northern analysis. Sequence comparison and Northern analysis proved that L1 is exactly the human ribosomal protein S19. It was present ubiquitously in 13 tissues tested but in various levels and even in different size. L1 was highly expressed in parotidean cystadenocarcinoma, pancreatic cancer and breast cancer examined. No mutations have been found in human uterine leiomyomas (n=6). CONCLUSIONS: hRPS19 overexpression might be a universal signal in rapid cell growth tissues.

DNA, Complementary↗
PubMed2005

DNA microarray data imputation and significance analysis of differential expression.

MOTIVATION: Significance analysis of differential expression in DNA microarray data is an important task. Much of the current research is focused on developing improved tests and software tools. The task is difficult not only owing to the high dimensionality of the data (number of genes), but also because of the often non-negligible presence of missing values. There is thus a great need to reliably impute these missing values prior to the statistical analyses. Many imputation methods have been developed for DNA microarray data, but their impact on statistical analyses has not been well studied. In this work we examine how missing values and their imputation affect significance analysis of differential expression. RESULTS: We develop a new imputation method (LinCmb) that is superior to the widely used methods in terms of normalized root mean squared error. Its estimates are the convex combinations of the estimates of existing methods. We find that LinCmb adapts to the structure of the data: If the data are heterogeneous or if there are few missing values, LinCmb puts more weight on local imputation methods; if the data are homogeneous or if there are many missing values, LinCmb puts more weight on global imputation methods. Thus, LinCmb is a useful tool to understand the merits of different imputation methods. We also demonstrate that missing values affect significance analysis. Two datasets, different amounts of missing values, different imputation methods, the standard t-test and the regularized t-test and ANOVA are employed in the simulations. We conclude that good imputation alleviates the impact of missing values and should be an integral part of microarray data analysis. The most competitive methods are LinCmb, GMC and BPCA. Popular imputation schemes such as SVD, row mean, and KNN all exhibit high variance and poor performance. The regularized t-test is less affected by missing values than the standard t-test. AVAILABILITY: Matlab code is available on request from the authors.

Algorithms↗
PubMed1999

cDNA representational difference analysis of differentially expressed cDNA sequences in human nasopharyngeal carcinoma.

OBJECTIVE: To search differentially expressed sequences correlated with pathogenesis of human nasopharyngeal carcinoma (NPC), including the candidates of tumor suppressor genes. METHODS: Representational difference analysis (RDA) was performed to isolate differentially expressed sequences between cDNA from normal human primary cultures of nasopharyngeal epithelial cells and cDNA from NPC cell line HNE1. The source of differentially expressed products were proved by Southern blot, Northern blot and in situ hybridization. The fragments were cloned with pGEM-T easy kit and sequenced by the chain termination reaction. RESULTS: Four differentially expressed cDNA fragments were isolated in the fourth subtractive hybridization using cDNA from normal human nasopharyngeal epithelial cells as tester amplicon and cDNA from NPC cell line HNE1 as driver amplicon by cDNA RDA. These differential cDNA fragments revealed that they really came from the tester amplicon and were not expressed or down-regulated in the NPC HNE1 cells. Some of the genes were expressed only in human nasopharyngeal epithelial cells but deleted or down-regulated in the biopsies of NPC. Of these obtained clones, some were the sequences of the human known genes including house-keeping genes, the others represented novel gene sequences. CONCLUSION: The differentially expressed products including the candidates of tumor-suppressor genes may be associated with the initiation of the NPC.

DNA, Complementary↗
PubMed1988

Analysis of F9 embryonal carcinoma cell lactosaminoglycans in relation to their differential expression during induction of differentiation.

Teratocarcinoma stem cells can be used to study the events related to early differentiation, and many cell surface changes have been described which correlate with the different stages of early embryogenesis. In this work we analyze the [3H]galactose-labeled glycopeptides derived from the mouse embryonal carcinoma cell line F9. We show that the high-molecular-weight glycopeptides typical of embryonal carcinoma cells are composed of two distinct molecular weight classes, namely H1 and H3, and that retinoic acid-induced differentiation determines a relative increase of the larger peak (H1) which is mainly due to a decrease in the expression of H3 species. We also show that, beside this decrease, there is a greater increase in the expression of lower-molecular-weight species. Furthermore, we present evidence that H1 and H3 species are polylactosaminoglycans N-linked to the peptidic backbone, and that induction of differentiation determines slight modifications in the structure of such species.

Amino Sugars↗
PubMed1998

Identification and cloning of differentially expressed genes by long-distance differential display.

Differential mRNA display (DD-PCR) amplifies short cDNAs (average size 100-350 bp), representing mainly the 3' untranslated regions (3' UTR) of transcripts. Sequencing of these cDNAs is predominantly uninformative for prediction of function and selection of clones for further analysis. Differential display of longer amplicons (0.5-2.0 kb) could enable isolation of cDNAs that encompass both 3' UTR and at least part of the 3' end of the coding region. The coding sequence information could facilitate selection of candidate clones for further analysis without the necessity of screening cDNA libraries. By combining DD-PCR protocols with long-distance PCR and using hot-start PCR with rTth DNA polymerase we have successfully amplified and comparatively displayed cDNAs ranging in size from 150 bp to 2 kb. Long-distance DD-PCR (LDD-PCR) has generated highly reproducible primer-specific patterns of cDNA fragments, as well as reproducible duplicate fingerprints, obtained from different RNA and cDNA samples. Sequencing and expression analyses of LDD-PCR clones have shown that LDD-PCR (a) enables nonredundant clone sampling, (b) generates many clones that encompass part of the coding region, and (c) samples both abundant and rare transcripts, approximately 60% of which are differentially expressed as confirmed by Northern analysis. Coupled with high-throughput cDNA sequencing and multiplex hybridization of cDNA microarrays for confirmation of differential expression, LDD-PCR could prove to be useful for simultaneous scanning of transcripts from multiple cDNA samples and faster selection of differentially expressed transcripts of interest.

Animals↗
PubMed1998

Detection of differentially expressed genes in primary tumor tissues using representational differences analysis coupled to microarray hybridization.

The identification of differential gene expressionbetween cells is a frequent goal in modern biological research. Here we demonstrate the coupling of representational difference analysis (RDA) of cDNA with microarray analysis of the output for high throughput screening. Two primary Ewing's sarcoma tissue samples with different biological behavior in vivo were compared by RDA: one which was metastatic and progressed rapidly; the other localized and successfully treated. A modified RDA protocol that minimizes the necessary starting material was employed. After a reduced number of subtractive rounds, the output of RDA was shotgun cloned into a plasmid vector. Inserts from individual colonies from the subtracted library were amplified with vector-specific primers and arrayed at high density on glass slides. The arrays were then hybridized with differentially fluorescently labeled starting amplicons from the two tissues and fluorescent signals were measured at each DNA spot. We show that the relative amounts of fluorescent signal correlate well with the abundance of fragments in the RDA amplicon and in the starting mRNA. In our system, we analyzed 192 products and 173 (90%) were appropriately detected as being >2-fold differentially expressed. Fifty unique, differentially expressed clones were identified. Therefore, the use of RDA essentially provides an enriched library of differentially expressed genes, while analysis of this library with microarrays allows rapid and reproducible screening of thousands of DNA molecules simultaneously. The coupling of these two techniques in this system resulted in a large pool of differentially expressed genes.

3T3 Cells↗
PubMed2004

The membrane proteases adams and hepsin are differentially expressed in renal cell carcinoma. Are they potential tumor markers?

PURPOSE: ADAMs (a disintegrin and metalloproteinases) as cell surface proteins with adhesion and protease activity, and hepsin as a transmembrane protease have important roles in many biological processes. We assessed the expression of various ADAMs and of hepsin in human renal cell carcinoma (RCC), and correlated expression with clinicopathological data. MATERIALS AND METHODS: mRNA expression of ADAM-8, 17, 19, 28, TS1 and TS2, and of hepsin was investigated in paired tissue samples from cancerous and noncancerous parts of the kidneys of 27 patients with RCC who underwent tumor nephrectomy. Measurements were performed by quantitative real-time reverse transcriptase-polymerase chain reaction on a LightCycler instrument (Roche Applied Science, Mannheim, Germany). The data were related to housekeeping gene porphobilinogen deaminase mRNA. RESULTS: All ADAMs except ADAM-TS1 were significantly higher but hepsin was less expressed (at least p <0.05) in cancerous vs matched noncancerous tissue. Expression was differentially related to tumor stage. ADAM-8 and ADAM-TS2 over expression as well as decreased hepsin expression were associated with shorter patient survival. The Cox proportional hazards regression model revealed that ADAM-TS2 was an independent prognostic factor for cancer related death. ADAM-8 was the best predictor of distant metastases. CONCLUSIONS: The differential expression of hepsin and ADAMs suggests early and late involvement of membrane proteases in the development of RCC. Their association with the clinical outcome illustrates their potential usefulness as biomarkers for RCC.

ADAM Proteins↗