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Morphological changes in tumour type after radiotherapy are accompanied by changes in gene expression profile but not in clinical behaviour.

The morphological features of neoplastic cells, combined with a stromal reaction, determine the presence of cancer at the microscopic level. Malignant tumours arise through a series of genetic alterations, but these do not entirely explain invasive and metastatic behaviour and correlate only weakly with morphological changes. In order to understand the relationship between the morphology of cancer tissue, gene expression, and clinical behaviour, a study of radiation-induced mucinous rectal carcinoma was performed. Short-term radiotherapy of rectal carcinoma results in an increased incidence of mucinous carcinoma. A cohort of rectal carcinomas (n = 1304), from patients who participated in a randomized radiotherapy trial, was evaluated for the presence and amount of a mucinous component. The results were compared with data from the pre-irradiation biopsies and revealed the presence of two distinct classes of mucinous carcinoma in the radiotherapy group, namely pre-existing (un-induced; n = 24) and induced mucinous carcinoma (n = 29). Clinical data, pathological parameters, and immunohistochemical data from these patients and their tumours showed that induced mucinous carcinomas were more comparable to typical adenocarcinomas than to pre-existing mucinous carcinomas. The prognosis of patients with induced mucinous carcinoma was significantly better than that of patients with pre-existing mucinous carcinomas (91.2% versus 39.3% recurrence-free interval at 2 years, p = 0.02). Gene expression profiles of the different groups of mucinous carcinomas and adenocarcinomas were analysed using Affymetrix Human Cancer Chips. Surprisingly, despite the difference in prognosis, the expression profile of radiation-induced mucinous carcinomas was very closely related to that of their un-induced counterparts. It is shown in the present study that radiation therapy of rectal cancer leads within a few days to substantial changes in both morphology and expression profile. However, the morphology of the pre-therapy biopsy predicts patient survival far better than post-therapy expression profiles. It is concluded that tumour morphology equates to expression profile, but that external factors might influence both, leading to sub-optimal prognostication.

Adenocarcinoma, Mucinous↗

Gene expression profile analysis in human hepatocellular carcinoma by cDNA microarray.

We performed gene expression profiling of normal and hepatocellular carcinoma (HCC) liver tissues using a high-density microarray that contained 3,063 human cDNA. The results of a microarray hybridization experiment from eight different HCC tissues were analyzed and classified by the Cluster program. Among these differentially-expressed genes, the galectin-3, serine/threonine kinase SGK, translation factor eIF-4A, -4B, -3, fibroblast growth factor receptor, and ribosomal protein L35A were up-regulated; the mRNAs of Nip3, decorin, and the insulin-like growth factor binding protein-3 were down-regulated in HCC. The differential expression of these genes was further confirmed by an RT-PCR analysis. In addition, our data suggest that the gene expression profile of HCC varies according to the histological types.

Carcinoma, Hepatocellular↗

[Exploration of gene expression profiles of CD(34)(+) hematopoietic stem/progenitor cells based on large scale sequencing].

OBJECTIVE: To set up a large scale sequencing system and explore the gene expression profiles of CD(34)(+) hematopoietic stem/progenitor cells (HSPCs). METHODS: CD(34)(+) cells were isolated from umbilical cord blood and subjected to cDNA library construction. A primary profile of gene expression in HSPCs was emerged by EST sequencing and bioinformatics analyzing. RESULTS: Among 9,866 ESTs thus obtained, 7 476 meaningful ESTs were clustered into 2,060 unique sequence species (USSs), representing 1,054 known gene species and 1,006 unknown gene fragments. The 1054 known genes were divided into 8 categories: (1) hematopoiesis associated: 73, (2) chromatin structure and cell division/apoptosis: 91, (3) signal transduction and receptors: 111, (4) cell structure/mobility: 48, (5) cell/organism defense/homeostasis: 41; (6) Gene expression (transcription, translation and modification): 265, (7) metabolism: 192; and (8) unclassified: 233. CONCLUSION: A gene expression profile including 1,054 known genes and 1006 new gene fragments of HSPCs was primarily obtained, which may lay a basis for the further study on the molecular mechanism of hematopoiesis regulation and provide candidates for new gene cloning.

Antigens, CD34↗

Temporal and spatial expression profile of the novel armadillo-related gene, Alex2, during testicular differentiation in the mouse embryo.

In a screen for transcripts differentially expressed during gonadal development in mouse embryos, we identified the novel armadillo-related gene, Alex2. The armadillo (arm) family of proteins share a 42 amino acid tandem repeat motif called the arm domain, through which they interact with different binding partners. These intracellular proteins are implicated in a variety of developmental processes, including cell proliferation, migration, maintenance of tissue integrity, and tumorigenesis. Alex2 is a member of a novel subgroup within the arm family, encoding a protein with a single arm domain and a putative transmembrane or signal sequence. Alex2 has a developmentally regulated expression profile during embryogenesis in the mouse. In the urogenital system, it is strongly expressed in the developing testis but is down-regulated during ovarian development. Alex2 expression is localized within the interstitial cell lineage of the developing testis, which gives rise to peritubular myoid, endothelial, and fetal Leydig cells. Alex2 is also expressed in the developing forebrain and somites and in dorsal root ganglia. In testicular cell lines, Alex2 fusion proteins localize to membrane structures within the cell. The expression profile of Alex2 suggests that it plays a role in the development of several tissues during embryogenesis, notably testicular differentiation. In the developing testis, its expression profile suggests that Alex2 has a role in specification or development of the interstitial cell lineage.

Amino Acid Sequence↗

Molecular identification of ERalpha-positive breast cancer cells by the expression profile of an intrinsic set of estrogen regulated genes.

Estrogens exert a key biological role in mammary gland epithelial cells and promote breast carcinogenesis and tumor progression. We recently identified a new large set of estrogen responsive genes from breast cancer (BC) cells by DNA microarray analysis of the gene expression profiles induced by 17beta-estradiol in ZR-75.1 and MCF-7 cells. The purpose of the present study was to test whether the expression pattern of hormone regulated genes from this set identifies estrogen receptor (ERalpha) positive, hormone responsive BC cells. To this aim, we carried out in silico metanalysis of ERalpha positive and ERalpha negative human BC cell line transcriptomes, focusing on two sets of 171 and 218 estrogen responsive genes, respectively. Results show that estrogen dependent gene activity in hormone responsive BC cells is significantly different from that of non-responsive cells and, alone, allows to discriminate these two cellular phenotypes. Indeed, we have identified 61 genes whose expression profile specifically marks ERalpha positive BC cells, suggesting that this gene set may be exploited for phenotypic characterization of breast tumors. This possibility was tested with data obtained by gene expression profiling of BC surgical samples, where the ERalpha positive phenotypes were highlighted by the expression profile of a subset of 27 such hormone responsive genes and four additional BC marker genes, not including ERs. These results provide direct evidence that the expression pattern of a limited number of estrogen responsive genes can be exploited to assess the estrogen signaling status of BC cells both in vitro and ex-vivo.

Biopsy↗

Gene expression profiling of mouse host response to Listeria monocytogenes infection.

The purpose of this study was to evaluate gene expression profiles in the liver and blood for prediction of infection severity from Listeria monocytogenes (LM). Mice were injected with medium broth (control) or a nonlethal or lethal dose of LM and sacrificed 6 h later. Gene expression changes were determined using Affymetrix MGU74Av2 GeneChips and confirmed by real-time polymerase chain reaction analysis. We identified discernable genes whose gene expression profiles can be used in pattern recognition to predict and classify samples in differently treated groups, with >or=90% accuracy in liver samples and 80% accuracy in blood at prediction; however, different genes were predictive in each tissue. Our results suggest that gene expression profiling in response to LM in mice may be able to distinguish samples in groups with varying severity of infection and provide information in finding molecular mechanisms and early biomarkers for subsequent conventional clinical endpoints.

Alanine Transaminase↗

Distinctive gene expression profiles associated with Hepatitis B virus x protein.

Hepatitis B virus (HBV) is a major risk factor for the development of hepatocellular carcinoma (HCC). HBV encodes the potentially oncogenic HBx protein, which mainly functions as a transcriptional co-activator involving in multiple gene deregulations. However, mechanisms underlying HBx-mediated oncogenicity remain unclear. To determine the role(s) of HBx in the early genesis of HCC, we utilized the NCI Oncochip microarray that contains 2208 human cDNA clones to examine the gene expression profiles in either freshly isolated normal primary adult human hepatocytes (Hhep) or an HCC cell line (SK-Hep-1) ecotopically expressing HBx via an adenoviral system. The gene expression profiles also were determined in liver samples from HBV-infected chronic active hepatitis patients when compared with normal liver samples. The microarray results were validated through Northern blot analysis of the expression of selected genes. Using reciprocally labeling hybridizations, scatterplot analysis of gene expression ratios in human primary hepatocytes expressing HBx demonstrates that microarrays are highly reproducible. The comparison of gene expression profiles between HBx-expressing primary hepatocytes and HBV-infected liver samples shows a consistent alteration of many cellular genes including a subset of oncogenes (such as c-myc and c-myb) and tumor suppressor genes (such as APC, p53, WAF1 and WT1). Furthermore, clustering algorithm analysis showed distinctive gene expression profiles in Hhep and SK-Hep-1 cells. Our findings are consistent with the hypothesis that the deregulation of cellular genes by oncogenic HBx may be an early event that favors hepatocyte proliferation during liver carcinogenesis.

Adult↗

Monitoring the expression profiles of doxorubicin-resistant K562 human leukemia cells by serial analysis of gene expression.

We examined the expression profiles of doxorubicin-resistant K562 cells by serial analysis of gene expression (SAGE) to identify novel and/or partially characterized genes that might be related to drug resistance in human leukemia. SAGE complementary DNA (cDNA) libraries were constructed from K562 and doxorubicin-resistant K562 (K562/ADM) cells, and concatamer sequences were analyzed with SAGE 2000 software. We used 9792 tags in the identification of 1076 different transcripts, 296 of which were similarly expressed in K562 and K562/ADM cells. There were 343 genes more actively expressed in K562/ADM than in parental K562 cells and 437 genes expressed less often in K562/ADM cells. K562/ADM cells showed increased expression of well-known genes, including the genes for spectrin beta, eukaryotic translation initiation factor 1A (EIF1A), RAD23 homolog B, laminin receptor 1, and polyA-, RAN-, and PAI-1 messenger RNA-binding proteins. K562/ADM cells showed decreased expression of the genes for fatty acid desaturase 1 (FADS1), hemoglobin epsilon 1, N-myristoyltransferase 1, hemoglobin alpha 2, NADH dehydrogenase Fe-S protein 6, heat shock 90-kDa protein, and karyopherin beta 1. Quantitative reverse transcription-polymerase chain reaction analysis confirmed the increased expression of EIF1A and the decreased expression of FADS1 in K562/ADM cells. Prior to this investigation, such differences in the expression of these genes in doxorubicin-resistant leukemia cells were unknown. Although we do not provide any evidence in the present report for the potential roles of these genes in drug resistance, SAGE may provide a perspective into our understanding of drug resistance in human leukemia that is different from that provided by cDNA microarray analysis.

Delta-5 Fatty Acid Desaturase↗

Identification and classification of differentially expressed genes in renal cell carcinoma by expression profiling on a global human 31,500-element cDNA array.

We investigated the changes in gene expression accompanying the development and progression of kidney cancer by use of 31,500-element complementary DNA arrays. We measured expression profiles for paired neoplastic and noncancerous renal epithelium samples from 37 individuals. Using an experimental design optimized for factoring out technological and biological noise, and an adapted statistical test, we found 1738 differentially expressed cDNAs with an expected number of six false positives. Functional annotation of these genes provided views of the changes in the activities of specific biological pathways in renal cancer. Cell adhesion, signal transduction, and nucleotide metabolism were among the biological processes with a large proportion of genes overexpressed in renal cell carcinoma. Down-regulated pathways in the kidney tumor cells included small molecule transport, ion homeostasis, and oxygen and radical metabolism. Our expression profiling data uncovered gene expression changes shared with other epithelial tumors, as well as a unique signature for renal cell carcinoma. [Expression data for the differentially expressed cDNAs are available as a Web supplement at http://www.dkfz-heidelberg.de/abt0840/whuber/rcc.]

Carcinoma, Renal Cell↗

Gene expression profiling reveals unique pathways associated with differential severity of lyme arthritis.

The murine model of Lyme disease provides a unique opportunity to study the localized host response to similar stimulus, Borrelia burgdorferi, in the joints of mice destined to develop severe arthritis (C3H) or mild disease (C57BL/6). Pathways associated with the response to infection and the development of Lyme arthritis were identified by global gene expression patterns using oligonucleotide microarrays. A robust induction of IFN-responsive genes was observed in severely arthritic C3H mice at 1 wk of infection, which was absent from mildly arthritic C57BL/6 mice. In contrast, infected C57BL/6 mice displayed a novel expression profile characterized by genes involved in epidermal differentiation and wound repair, which were decreased in the joints of C3H mice. These expression patterns were associated with disease state rather than inherent differences between C3H and C57BL/6 mice, because C57BL/6-IL-10(-/-) mice infected with B. burgdorferi develop more severe arthritis than C57BL/6 mice and displayed an early gene expression profile similar to C3H mice. Gene expression profiles at 2 and 4 wk postinfection revealed a common response of all strains that was likely to be important for the host defense to B. burgdorferi and mediated by NF-kappaB-dependent signaling. The gene expression profiles identified in this study add to the current understanding of the host response to B. burgdorferi and identify two novel pathways that may be involved in regulating the severity of Lyme arthritis.

Animals↗

Global gene expression profiling of preimplantation embryos.

Preimplantation development is marked by four major events: the transition of maternal transcripts to zygotic transcripts, compaction, the first lineage differentiation into inner cell mass and trophectoderm, and implantation. The scarcity of the materials of preimplantation embryos, both in size (diameter < 100 microm) and in quantity (only a few to tens of oocytes from each ovulation), has hampered molecular analysis of preimplantation embryos. Recent progress in RNA amplification methods and microarray platforms, including genes unique to preimplantation embryos, allow us to apply global gene expression profiling to the study of preimplantation embryos. Our gene expression profiling during preimplantation development revealed the distinctive patterns of maternal RNA degradation and embryonic gene activation, including two major transient waves of de novo transcription. The first wave corresponds to zygotic genome activation (ZGA). The second wave, mid-preimplantation gene activation (MGA), contributes dramatic morphological changes during late preimplantation development. Further expression profiling of embryos treated with inhibitors of transcription or translation revealed that the translation of maternal RNA is required for the initiation of ZGA, suggesting a cascade of gene activation from maternal RNA/protein sets to ZGA gene sets and thence to MGA gene sets. To date, several reports of microarray experiments using mouse and human preimplantation embryos have been published. The identification of a large number of genes and multiple signaling pathways involved at each developmental stage by such global gene expression profiling accelerates understanding of molecular mechanisms underlining totipotency/pluripotency and programs of early mammalian development.

Animals↗

Gene expression profile induced by BCNU in human glioma cell lines with differential MGMT expression.

Chemotherapy with the alkylating agent BCNU (1,3-bis (2-chloroethyl)-1-nitrosourea) is the most commonly used chemotherapeutic agent for gliomas. However, the usefulness of this agent is limited because tumor cell resistance to BCNU is frequently found in clinical brain tumor therapy. The O6-methylguanine-DNA methyltransferase protein (MGMT) reverses alkylation at the O6 position of guanine and we have reported the role of MGMT in the response of brain tumors to alkylating agents. However, the different mechanisms underlying the patterns related to MGMT remain unclear. To better understand the molecular mechanism by which BCNU exerts its effect in glioma cell lines according MGMT expression, we used microarray technology to interrogate 3800 known genes and determine the gene expression profiles altered by BCNU treatment. Our results showed that treatment with BCNU alters the expression of a diverse group of genes in a time-dependent manner. A subset of gene changes was found common in both glioma cell lines and other subset is specific of each cell line. After 24 h of BCNU treatment, up-regulation of transcription factors involved in the nucleation of both RNA polymerase II and III transcription initiation complexes was reported. Interestingly, BCNU promoted the expression of actin-dependent regulators of chromatin. Similar effects were found with higher BCNU doses in MGMT+ cell line showing a similar mechanism that in MGMT-deficient cell with standard doses. Our data suggest that human glioma cell lines treated with BCNU, independently of MGMT expression, show changes in the expression of cell cycle and survival-related genes interfering the transcription mechanisms and the chromatin regulation.

Antineoplastic Agents, Alkylating↗

Functional and genomic implications of global gene expression profiles in cell lines from human hepatocellular cancer.

Global gene expression profiles in cancer have impacted both classification of tumors and definition of molecular pathways in neoplasia. To explore the possibility of employing human tumor cell lines to obtain information on the functional genomics of the early stages of tumorigenesis, we have characterized variation in gene-expression patterns in a cytogenetically well-defined series of cell lines derived from human hepatocellular carcinoma (HCC). Microarrays containing 6,720 sequence-verified human cDNAs were used in this study. Nineteen well-characterized HCC cell lines were analyzed, and a nontumorigenic liver-derived epithelial cell line (Chang) was used as a reference. Each sample was examined at least twice by switching fluorescent dyes, Cy-5 and Cy-3, and average values of 2 experiments on each sample were used for further analysis. Analysis of the clustered data revealed 2 distinctive subtypes of gene-expression patterns among the 19 cell lines, suggesting a degree of heterogeneity among the gene-expression profiles of cell lines. Remarkably, expression of alpha-fetoprotein (AFP) was highly correlated with the molecular subtypes of HCC. Although the 3 most distinctive gene-expression modules represented the signatures of 2 different subgroups of HCC, most of the cell lines shared many coexpressed genes. However, sets of coexpressed genes that are specific for the subtypes of HCC were identified. Furthermore, our results indicate that the comparison between gene-expression patterns and structural alterations in chromosomes is potentially useful in identifying genes critical in early stages of tumorigenesis. In conclusion, these results not only identified unrecognized subtypes of HCC, but also provided potential molecular markers for each subtype that can be useful for diagnostic and/or therapeutic purposes.

Carcinoma, Hepatocellular↗

Molecular characterization of human meningiomas by gene expression profiling using high-density oligonucleotide microarrays.

Meningiomas are common central nervous system neoplasms that exhibit remarkably diverse histopathology and biological behavior. Compared to astrocytomas, the most common central nervous system tumor, little is known about the molecular pathways critical for meningioma tumor formation and malignant progression. As an initial step toward characterizing the genetic basis of meningioma pathogenesis, we assessed cancer-related gene expression profiles of nonneoplastic leptomeningeal specimens and human meningiomas of varying World Health Organization (WHO) grade using high-density oligonucleotide microarrays. Although expression profile differences between nonneoplastic and meningioma specimens were readily discernible, the expression profile of a subset of genes could also distinguish WHO grade I from WHO grades II and III tumors. Altered expression levels of several genes identified in this study have been previously noted in meningiomas (eg, growth hormone receptor, IGFBP-7, endothelin receptor A, IGF2). However, we also identified a number of novel genes whose expression was associated with WHO grade and was confirmed by reverse transcriptase-polymerase chain reaction in a larger, independent set of meningeal tumors (n = 47). This report represents the first gene expression profiling studies of meningiomas and identifies some initial candidate genes that may provide further insights into the genetic basis for meningioma pathogenesis.

Carrier Proteins↗

Microarray expression profiling: capturing a genome-wide portrait of the transcriptome.

The bacterial transcriptome is a dynamic entity that reflects the organism's immediate, ongoing and genome-wide response to its environment. Microarray expression profiling provides a comprehensive portrait of the transcriptional world enabling us to view the organism as a 'system' that is more than the sum of its parts. The vigilance of microorganisms to environmental change, the alacrity of the transcriptional response, the short half-life of bacterial mRNA and the genome-scale nature of the investigation collectively explain the power of this method. These same features pose the most significant experimental design and execution issues which, unless surmounted, predictably generate a distorted image of the transcriptome. Conversely, the expression profile of a properly conceived and conducted microarray experiment can be used for hypothesis testing: disclosure of the metabolic and biosynthetic pathways that underlie adaptation of the organism to chang-ing conditions of growth; the identification of co-ordinately regulated genes; the regulatory circuits and signal transduction systems that mediate the adaptive response; and temporal features of developmental programmes. The study of bacterial pathogenesis by microarray expression profiling poses special challenges and opportunities. Although the technical hurdles are many, obtaining expression profiles of an organism growing in tissue will probably reveal strategies for growth and survival in the host's microenvironment. Identifying these colonization strategies and their cognate expression patterns involves a 'deconstruction' process that combines bioinformatics analysis and in vitro DNA array experimentation.

Bacteria↗

Comparing expression profiles of genes with similar promoter regions.

MOTIVATION: Gene regulatory elements are often predicted by seeking common sequences in the promoter regions of genes that are clustered together based on their expression profiles. We consider the problem in the opposite direction: we seek to find the genes that have similar promoter regions and determine the extent to which these genes have similar expression profiles. RESULTS: We use the data sets from experiments on Saccharomyces cerevisiae. Our similarity measure for the promoter regions is based on the set of common mapped or putative transcription factor binding sites and other regulatory elements in the upstream region of the genes, as contained in the Saccharomyces cerevisiae Promoter Database. We pair up the genes with high similarity scores and compare their expression levels in time-course experiment data. We find that genes with similar promoter regions on the average have significantly higher correlation, but it can vary widely depending on the genes. This confirms that the presence of similar regulatory elements often does not correspond to similarity in expression profiles and indicates that finding transcription factor binding sites or other regulatory elements starting with the expression patterns may be limited in many cases. Regardless of the correlation, the degree to which the profiles agree under different experimental conditions can be examined to derive hypotheses concerning the role of common regulatory elements. Overall, we find that considering the relationship between the promoter regions and the expression profiles starting with the regulatory elements is a difficult but useful process that can provide valuable insights.

Databases, Nucleic Acid↗

Expression profiling of GABAA receptor beta-subunits in the rat retina.

This study profiled the expression of the family of GABAA receptor beta-subunits in the adult rat retina. Using a combination of reverse transcriptase reaction followed by polymerase chain reaction (RT-PCR) with gene-specific primers, the expression of mRNAs encoding the beta 1, beta 2, and beta 3 subunits was first examined in the intact retina and then in separated retinal nuclear layers. However, it was found that a critical analysis had to be carried out at the level of the single cell in order to resolve the differential patterns of expression among the retinal cell types. When cells were isolated and identified following acute dissociation, RT-PCR revealed that individual rod photoreceptor cells expressed consistently the beta 1 and beta 2 messages while the bipolar cells expressed the beta 1 and beta 3 messages. Ganglion cells displayed considerable variability in beta-subunit expression, perhaps reflecting their functional and morphological heterogeneity in the retina. In contrast, the nonneuronal Mueller cells did not express any of the beta-subunit messages. These results indicate that the expression of GABAA receptor subunits is cell-type dependent. Furthermore, as the expression of other families of GABAA receptor subunits are profiled and the patterns of subunit assembly are better understood, our results raise the possibility that GABAA receptors with different subunit compositions can be expected to be coexpressed within a single retinal neuron.

Animals↗

Gene expression profiles in mouse lung tissue after administration of two cationic polymers used for nonviral gene delivery.

PURPOSE: This study compared gene expression profiles in mouse lungs after administration of the cationic polymers polyethyleneimine (PEI) or chitosan alone or formulated with a luciferase reporter plasmid (PEI-pLuc, chitosan-pLuc). METHODS: The polymers and formulations were administered intratracheally to Balb/c mice at doses judged to be nontoxic according to intracellular dehydrogenase activity and tissue morphology. RNA was isolated from the lungs 24 or 72 h after administration, and a dedicated stress and toxicology cDNA array was used to monitor the in vivo response to the gene delivery system in the lung tissue. RESULTS: The gene expression profiles differed between the PEI and chitosan groups with regard to both the total number and the type of expressed genes. Chitosan-pLuc upregulated genes that protect the cell from oxidative stress and inflammation, such as heme oxygenase-1 and catalase, whereas PEI-pLuc upregulated genes involved in inflammatory processes, such as the cyclooxygenases 1 and 2, indicating possible involvement in the development of adverse reactions. However, both polymers activated genes involved in reaction to stress, such as DNA damage repair. Furthermore, in the PEI group, chaperone genes and members of the p38 mitogen-activated protein kinase pathway were also upregulated, suggesting a possible explanation for the better performance of PEI in gene delivery systems. CONCLUSIONS: The results indicate that gene expression profiling is a useful and sensitive tool for the evaluation of tissue responses after administration of polymers or gene delivery systems. The results also suggest a possible explanation for the differences in gene delivery performance between the two polymers in gene delivery systems.

Animals↗