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PubMed2005

Gene expression profiling using RNA extracted from whole blood: technologies and clinical applications.

Gene expression profiling promises to provide an insight into normal biologic and pathologic processes with the hope of predicting disease outcome or indicating individualized courses of therapy. The entire process for gene expression profiling from clinical samples consists of sample collection, transport and storage of clinical sample, isolation of nucleic acids, enzymatic modification of nucleic acids, detection and data analysis. All steps exert an influence on the quality, accuracy and reliability of the final result. A standardization of the entire process from sample collection to nucleic acid analysis is therefore required in order to achieve reliable gene expression results. After providing a general overview of technologies for gene expression profiling and respective caveats, this review will focus on clinical applications of blood sample profiling.

Acute Disease↗

PubMed2002

Molecular characterization of breast cancer cell lines by expression profiling.

PURPOSE: Gene expression patterns provide detailed insights into cellular regulation that reflect minor differences of cellular capacity not accessible by standard descriptions of the cellular phenotype or origin. METHODS: To identify fundamental differences and similarities we analyzed the gene expression patterns of four breast cancer cell lines: MCF-7, SK-BR-3, T-47D, and BT-474. RESULTS: Although only a small subset of genes (597) is represented on the Atlas-cDNA-Array (Clontech) used, clear differences in the expression of a number of genes could be detected. For example, unique high levels of expressions were found for the HLH-protein ID-1 (MCF-7) and the receptor tyrosine kinase erbB2 (SK-BR-3 and T-47D). Most genes analyzed were expressed at comparable levels in all cell lines studied. CONCLUSIONS: For interpretation of the results sets of genes that show similar variation of expression among the cells were grouped together. Furthermore, our analysis allows the assignment of similarity values that lead to a relation profile of the cell lines. How these results correlate with known biological properties of the cell lines is discussed. Additionally, we demonstrate that results obtained by cDNA-Array hybridization for expression of the ErbB receptor family correlate well with competitive RT-PCR, thus confirming the reliability of the cDNA-Array analysis.

Breast Neoplasms↗

PubMed2003

Expression profiling and characterization of 4200 genes cloned from primary neuroblastomas: identification of 305 genes differentially expressed between favorable and unfavorable subsets.

Neuroblastoma (NBL), one of the most common childhood solid tumors, has a distinct nature in different prognostic subgroups: NBL in patients under 1 year of age usually regresses spontaneously, whereas that in patients over 1 year of age often grows aggressively and eventually kills the patient. To understand the molecular mechanism of biology and tumorigenesis of NBL, we decided to perform a comprehensive approach to unveil the gene expression profiles among the NBL subsets. We constructed the subset-specific oligo-capping cDNA libraries from the primary NBL tissues with favorable (F: stage 1, high expression of TrkA and a single copy of MYCN) and unfavorable (UF: stage 3 or 4, decreased expression of TrkA and MYCN amplification) characteristics and randomly cloned 4654 cDNAs. Among 4243 cDNAs sequenced successfully, 1799 (42.4%) were the genes with unknown function. Excluding the housekeeping genes, an expression profile of each subset was extremely different. To determine the genes expressed differentially between F and UF subsets, we performed semiquantitative reverse transcriptase (RT)-PCR for each of the 1842 independent genes using RNA obtained from 16 F and 16 UF NBLs as template. This revealed that 278 genes were highly expressed in the F subset as compared to the UF one, while, surprisingly, only 27 genes were expressed at higher levels in the UF rather than the F subset. These differentially expressed genes included 194 genes with unknown function. Many of the genes expressed at high levels in the F subset were related to catecholamine biosynthesis, small GTPases, synapse formation, synaptic vesicle transport, and transcription factors regulating differentiation of the neural crest-derived cells. On the other hand, the genes expressed at high levels in the UF subset included transcription factors and/or receptors that might regulate neuronal growth and differentiation. The chromosomal mapping of those genes showed some clusters. Thus, our mass-identification and characterization of the differentially expressed genes between the subsets may become a powerful tool for finding the important genes of NBL as well as developing new diagnostic and therapeutic strategies against aggressive NBL.

Chromosome Mapping↗

PubMed2001

Identification of differentially expressed genes in hepatocellular carcinoma and metastatic liver tumors by oligonucleotide expression profiling.

BACKGROUND: The characterization of differentially expressed genes between cancerous and normal tissues is an important step in the understanding of tumorigenesis. Global gene expression profiling with microarrays has now offered a powerful tool to measure the changes of thousands of genes in any carcinoma tissues in an effort to identify these key disease-related genes. To compare the gene expression of a primary liver carcinoma, metastatic carcinoma to the liver, and normal liver, the authors analyzed tissue from six primary hepatocellular carcinomas (HCCs), five colorectal adenocarcinoma metastases to the liver, and eight normal livers. METHODS: Samples were processed from total RNA to fragmented cRNA and hybridized onto Affymetrix GeneChip(R) expression arrays. Analyses were performed to determine the consensus pattern of gene expression for primary liver carcinoma, metastatic liver carcinoma, and normal liver tissue and their changes in expression level. RESULTS: In hepatocellular carcinoma, 842 genes were overexpressed, and 393 genes were underexpressed in comparison with genes of normal liver tissue. Of note, 7 of the 20 most increased identified known genes previously have been associated with liver carcinoma or other types of cancers. The 13 additional identified genes until now have not previously shown strong association with cancers. Furthermore, the authors identified 42 genes and 24 expressed sequence tags that are expressed at a significant level in both HCC and metastastic tumors, presenting a list of marker genes indicative of cancerous liver tissue. CONCLUSIONS: In this study, genes that can be involved in the production of and maintenance of hepatic carcinomas were identified. These data offer new insight into genes that are potentially important in the pathogenesis of liver carcinoma, as well as additional targets for new strategies for cancer therapy and treatment.

Adult↗

PubMed2006

[The down regulation of VEGF gene expression may link to change the expression profile of genes in leukemia cell line K562].

OBJECTIVE: To explore the potential effects of anti-VEGF hairpin ribozyme gene to gene expression profiles in leukemia cell line K562. METHODS: The lipofectamine mediation was used to transfect the recombinant eukaryotic expression plasmid (pcDNA3-RZ) containing anti-VEGF hairpin ribozyme gene and the non-recombinant vector as control into K562 cells. And the positive clones were screened by G418. Ribozyme gene in K562 cells was confirmed by PCR. Fluorescent real time reverse transcription-PCR(RT-PCR) and Western blotting were employed to detect the expression of VEGF mRNA and protein in leukemia cells. cDNA microarray was used to explore the alteration of gene expression profiles when decreasing VEGF gene expression in leukemia cells. Expression of PCNA and GSN genes were verified by semi-quantitative RT-PCR. RESULTS: The pcDNA3-RZ and pcDNA3 had been transfected into the human leukemia cell line K562 and positive clones been screened by G418. Stable expression of the ribozyme gene in K562 cells was confirmed by PCR. The level of VEGF mRNA and protein decreased dramatically in K562-RZ cells when compared with K562 or K562-PC (K562 cell transfected with empty vector) cells. The gene expression profiles were changed by transfection of anti-VEGF hairpin ribozyme gene into K562 cells. Among 4096 gene clones on the microarray, 191 (4.86%) genes were detected to have the marked changes with 104 down-regulated and 87 up-regulated, that were functionally related to cell cycle progression, gene replication, metabolism, cell apoptosis, cell signal transduction, and oncogenes etc. An increased expression of GSN gene and a decreased expression of PCNA gene in K562/RZ cells have been detected by RT-PCR. CONCLUSION: Down-regulation of VEGF gene by introducing anti-VEGF hairpin ribozyme gene can alter the gene expression profiles in K562 cells, leading to change of cell growth, differentiation and apoptosis in K562/RZ cells.

Down-Regulation↗

PubMed2004

Prediction of response to neoadjuvant chemotherapy for osteosarcoma by gene-expression profiles.

To establish a method for predicting the response to chemotherapy for osteosarcoma (OS), we performed expression profile analysis using cDNA microarray consisting of 23,040 genes. Hierarchical clustering based on the expression profiles of 19 biopsy samples of OS demonstrated two major clusters, one of which consisted exclusively of typical OS, i.e. conventional central OS in long bone of patients in the second decade. A set of genes was identified to characterize this subgroup, some of which have previously indicated some relation to carcinogenesis. Thirteen of the 19 patients were treated with an identical protocol of chemotherapy containing doxorubicin, cis-platinum and ifosfamide, and histological examination of resected specimens after operation classified 6 cases as responder and 7 as non-responder. A comparison of expression profiles of these two groups identified 60 genes whose expression levels were likely to be correlated with the response to chemotherapy (P<0.008). A drug response scoring (DRS) system was developed based on the expression levels of these genes, which proved to be applicable to predict the response to chemotherapy irrespective for the subclassification of OS. The reliability of the DRS system was further confirmed by testing additional 5 OS cases. These results indicated that scoring system based on gene-expression profiles might be useful to predict the response to chemotherapy for OS.

Adolescent↗

PubMed2003

Gene expression profile changes in NB4 cells induced by arsenic trioxide.

AIM: To investigate the gene expression profiles of acute promyelocytic leukemia (APL) cell line NB4 treated with arsenic trioxide (As2O3) using cDNA microarray. METHODS: Two cDNA probes were prepared through reverse transcription from mRNA of NB4 cells treated with or without arsenic trioxide. The probes were labeled with Cy3 and Cy5 fluorescence dyes individually, hybridized with cDNA microarray representing 1003 different human genes, and their fluorescent intensities were scanned. The genes were screened through the analysis of the difference in two gene expression profiles. RESULTS: The analysis of gene expression profiles indicated that after the treatment of arsenic trioxide (0.5 micromol/L) 3 genes were up-regulated, among which, PSMB6 gene was involved in proteasome degradation pathway, and 18 genes related to RNA processing, protein synthesis, and signal transduction were down-regulated. CONCLUSION: PSMB6 and ITGB1 genes may be related to the differentiation and/or apoptosis of NB4 cells induced by As2O3.

Antineoplastic Agents↗

PubMed2003

Microarray-based gene expression profiles of allograft rejection and immunosuppression in the rat heart transplantation model.

BACKGROUND: Gene expression profiling has the potential to produce new insights into complex biologic systems. To test the value of complement DNA arrays in identifying pathways involved in organ transplant rejection, we examined the gene expression profiles of rat heart allografts from recipients treated with or without immunosuppression to prevent acute allograft rejection. METHODS: Heterotopic heart transplantation was performed using ACI or Lewis donors and Lewis recipients. Recipients were treated with tacrolimus (Tac) or cyclosporine (CsA) at the equivalent effective doses, and graft hearts were harvested on days 3, 5, and 7. A commercial microarray was used to measure gene expression levels of 588 genes in day 5 grafts. Selected genes were analyzed by reverse transcriptase-polymerase chain reaction. RESULTS: The expression levels of 118 genes were perturbed in the untreated allograft in comparison with the isograft control, of which 77 genes were categorized as candidate genes for Tac- or CsA-mediated immunosuppression or both, and 41 as genes associated with other pathways. Among the 77 candidate genes, 55 genes shared the same response to suppression by both drugs, including inducible nitric oxide synthase, interferon-gamma, and interferon regulatory factor 1. Drug-specific effects were observed in 22 genes: Fourteen genes were exclusively reversed by Tac and eight by CsA. CONCLUSIONS: Gene expression profiling reveals a large variety of genes affected during acute rejection, indicating that multiple metabolic pathways, including immune and nonimmune responses, are involved in the local graft rejection events. The differences and similarities of the gene expression profiles relative to the two immunosuppressants may provide more detailed therapeutic approaches for optimal immunosuppression.

Animals↗

PubMedDate not supplied

Alterations in gene expression profiles and the DNA-damage response in ionizing radiation-exposed TK6 cells.

Identifying genes that are differentially expressed in response to DNA damage may help elucidate markers for genetic damage and provide insight into the cellular responses to specific genotoxic agents. We utilized cDNA microarrays to develop gene expression profiles for ionizing radiation-exposed human lymphoblastoid TK6 cells. In order to relate changes in the expression profiles to biological responses, the effects of ionizing radiation on cell viability, cloning efficiency, and micronucleus formation were measured. TK6 cells were exposed to 0.5, 1, 5, 10, and 20 Gy ionizing radiation and cultured for 4 or 24 hr. A significant (P < 0.0001) decrease in cloning efficiency was observed at all doses at 4 and 24 hr after exposure. Flow cytometry revealed significant decreases in cell viability at 24 hr in cells exposed to 5 (P < 0.001), 10 (P < 0.0001), and 20 Gy (P < 0.0001). An increase in micronucleus frequency occurred at both 4 and 24 hr at 0.5 and 1 Gy; however, insufficient binucleated cells were present for analysis at the higher doses. Gene expression profiles were developed from mRNA isolated from cells exposed to 5, 10, and 20 Gy using a 350 gene human cDNA array platform. Overall, more genes were differentially expressed at 24-hr than at the 4-hr time point. The genes upregulated (> 1.5-fold) or downregulated (< 0.67-fold) at 4 hr were those primarily involved in the cessation of the cell cycle, cellular detoxification pathways, DNA repair, and apoptosis. At 24 hr, glutathione-associated genes were induced in addition to genes involved in apoptosis. Genes involved in cell cycle progression and mitosis were downregulated at 24 hr. Real-time quantitative PCR was used to confirm the microarray results and to evaluate expression levels of selected genes at the low doses (0.5 and 1.0 Gy). The expression profiles reflect the cellular and molecular responses to ionizing radiation related to the recognition of DNA damage, a halt in progression through the cell cycle, activation of DNA-repair pathways, and the promotion of apoptosis.

Analysis of Variance↗

PubMed2006

Gene expression profile changes correlating with radioresistance in human cell lines.

PURPOSE: To identify gene expression profiles specific to radioresistance of human cells. METHODS AND MATERIALS: Global gene expression profiles of a total of 15 tumor and normal fibroblast cell lines were analyzed using DNA microarrays and statistical clustering methods. Initially, six of the cell lines were categorized into radioresistant (RG) or nonradioresistant (NRG) groups according to the radiation dose required to reduce their survival to 10% (D10). Genes for which expression was specific to each group at 1 or 3 h after irradiation were identified using statistical procedures including analysis of variance and a two-dimensional hierarchical clustering method. The remaining nine cell lines were subjected to the k-nearest neighbor pattern classification. RESULTS: The nine test cell lines were successfully classified by their D10 value using 46 and 44 genes for which transcription levels had significantly changed at 1 and 3 h after irradiation, respectively. Of these genes, 25 showed altered expression at both time points in the NRG or RG, but independently were unable to classify the test cell lines. CONCLUSIONS: Radioresistant cell lines analyzed in this study showed certain radiation-induced changes in gene expression profiles that are different from the profile changes of the more-sensitive cell lines.

Cell Line, Tumor↗

PubMed2003

High-throughput analysis of total RNA expression profiles by capillary gel electrophoresis.

We studied the expression profiles of total RNA in three types of human tissues. Reverse transcription was performed on total RNA samples extracted from kidney, normal breast, and breast tumor. Then, fluorophor-labeled cDNAs were synthesized by using random hexamers as primers for analysis by capillary gel electrophoresis. By overlaying the electropherograms and by performing multivariate analysis, we found that the RNA patterns were significantly different among the tissue samples. The protocol utilizes only submicroliter volumes of PCR products and is compatible with multiple capillary DNA sequencing instruments. These CE-based RNA patterns provide an alternative to RNA microarrays for expression profiling for understanding the molecular basis of disease.

Breast↗

PubMed2002

Expression profiling and comparative sequence derived insights into lipid metabolism.

Expression profiling and genomic DNA sequence comparisons are increasingly being applied to the identification and analysis of the genes that are involved in lipid metabolism. Not only has genome-wide expression profiling aided in the identification of novel genes that are involved in important processes in lipid metabolism such as sterol efflux, but also the utilization of information from these studies has added to our understanding of the regulation of pathways that participate in the process. Coupled with these gene expression studies, cross-species comparison (a technique used to search for sequences that are conserved through evolution) has proven to be a powerful tool to identify important noncoding regulatory sequences and novel genes that are relevant to lipid biology. An example of the value of this approach was the recent chance discovery of a new apolipoprotein gene (that which encodes apolipoprotein AV) that has dramatic effects on triglyceride metabolism in mice and humans.

Animals↗

PubMed2005

Gene expression profile of cytokines and chemokines in microdissected primary Hodgkin and Reed-Sternberg (HRS) cells: high expression of interleukin-11 receptor alpha.

We microdissected Hodgkin and Reed-Sternberg (HRS) cells from 14 Hodgkin's lymphoma tissue samples (nodular sclerosis = 5; mixed cellularity = 9), and after isolation and amplification of mRNA, analyzed the expression profile of 140 genes of chemokines, cytokines and their receptors by cDNA microarray methods. We also compared the profile with those of germinal center (GC) cells in reactive lymphadenitis. Unsupervised clustering revealed a relatively homogeneous expression profile in HRS cells. HRS cells tended to express mainly Th2 T cell-associated molecules rather than those of Th1, compared with GC cells. Interleukin-11 receptor alpha (IL-11Ralpha), a previously unknown HRS cell-specific gene, was detected in addition to known genes. Immunohistochemical staining confirmed the expression of IL-11Ralpha at the protein level. In contrast, only few cases were positive for IL-11Ralpha in B cell lymphoma, diffuse large cell lymphoma and follicular lymphoma. This is the first analysis report of tissue HRS cells with cDNA microarray technique.

Adolescent↗

PubMed2002

Expression profiling to predict outcome in breast cancer: the influence of sample selection.

Gene expression profiling of tumors using DNA microarrays is a promising method for predicting prognosis and treatment response in cancer patients. It was recently reported that expression profiles of sporadic breast cancers could be used to predict disease recurrence better than currently available clinical and histopathological prognostic factors. Having observed an overlap in those data between the genes that predict outcome and those that predict estrogen receptor-alpha status, we examined their predictive power in an independent data set. We conclude that it may be important to define prognostic expression profiles separately for estrogen receptor-alpha-positive and estrogen receptor-alpha-negative tumors.

Breast Neoplasms↗

PubMed1996

A gene expression profile of human corneal epithelium and the isolation of human keratin 12 cDNA.

PURPOSE: To describe the quantitative and qualitative aspects of gene expression in human corneal epithelium and to discover novel cornea-specific genes. METHODS: A 3'-directed cDNA library was constructed with messenger RNA prepared from normal human corneal epithelial cells, and inserts in 1069 randomly chosen clones were sequenced. These sequences were compared with each other to determine the frequency of appearance and were searched against GenBank for identification. The resultant expression profile, a list of gene species and their recurrences, reflected the composition of mRNA in the cornea. Recurrently appearing sequences, representing abundant transcripts, were compared with sequences in expression profiles obtained from seven other tissues and from those in dbEST to discover cornea-specific genes. RESULTS: The expression profile of human corneal epithelium showed that the most abundant transcript in this tissue was that for apolipoprotein J. Altogether 62 genes were suggested to be very active, including calcyclin, alpha-enolase, keratin 3, connexin 43, and 12 novel genes. The expression of four of these 12 novel genes seemed to be limited to cornea because they were not found in seven other expression profiles nor in dbEST. Full-length cDNA corresponding to one of these (GS8025), isolated from a separately made cDNA library, contained open reading frame highly homologous to mouse keratin 12, which is known to be cornea specific. CONCLUSIONS: An expression profile of corneal epithelium provides probes to monitor physiological and pathologic conditions of this tissue in terms of gene expression. Furthermore, by comparing this profile with those of other tissues, probes to isolate genes uniquely transcribed in corneal epithelium are determined. These genes are assumed to carry unique functions for this tissue and are candidate genes for inherited diseases that manifest only in cornea. As an example, human cornea-specific keratin was isolated, and partial cDNA sequences for three more cornea-specific genes were presented.

Adult↗

PubMed2006

Differential gene expression profiling in genetic and multifactorial cardiovascular diseases.

Gene expression profiling by microarray technologies has been successfully applied to study the transcriptional changes that occur in tissues such as heart, vessels and blood cells in different cardiovascular disorders. Such studies have been performed in human cardiovascular syndromes and in animal models with the aim of unraveling the complex molecular pictures underlying human pathophysiology. As already observed in cancer research, gene expression studies in humans may provide a finer molecular classification of patients with cardiovascular diseases and indicate new markers useful for prognostic and therapeutic strategies. In this paper, we present the findings obtained with microarray platforms to explore transcriptome alterations in cardiovascular diseases. To describe the potential of global expression profiling approach in this field, we have chosen to review the genomic findings obtained in some classic heart diseases with genetic transmission such as hyperthrophic cardiomyopathy and Fabry disease, together with findings obtained in common multifactorial cardiovascular disorders such as heart failure, atherosclerosis and infarction. Wherever feasible, we present the results obtained in patients together with those obtained in the corresponding animal and cellular models.

Animals↗

PubMed2002

Nuclei and subnuclei gene expression profiling in mammalian brain.

Information on the neuroanatomical expression of a given gene is critical to understanding its function in the central nervous system. The integration of laser capture microdissection (LCM), T7-based RNA amplification and cDNA microarrays allows for this information to be simultaneously generated for thousands of genes. To validate this integrative approach, we catalogued the gene expression profiles of seven rat brain nuclei or subnuclei. A hundred cells from the following seven brain nuclei were analyzed: locus coeruleus (LC), dorsal raphe nucleus (DR), parvocellular division (PA) and magnocellular division (MG) of the hypothalamic paraventricular nucleus (PVN) and CA1, CA3 and dentate gyrus (DG) divisions of the hippocampal formation. Of the 2145 genes investigated, 1402 genes (65%) gave a hybridization signal statistically different from the background level that was defined by non-specific hybridizations to 15 different plant genes. Validation of our microarray data on four arbitrarily selected genes was confirmed by Real-Time PCR. Previous research showing expression patterns of 'signature' genes (n=17) for specific brain nuclei are consistent with our findings. For example, as previously shown, enriched mRNA expression encoding the serotonin transporter or tyrosine hydroxylase was found in DR and LC cells, respectively. Interestingly, expression of the serotonin 5-HT(2B) receptor mRNA was also found in DR cells. We confirmed this new finding by in-situ hybridization. The hierarchical clustering analysis of gene expression shows that the two divisions of the PVN (PA and MG) are closely related to each other, as well as the three regions of the hippocampal formation (CA1, CA3 and DG), which also showed similar gene expression profiles. This study demonstrates the importance, feasibility and utility of cellular brain nuclei profiling.

Animals↗

PubMed2002

Expression profile of active genes in the human pituitary gland.

To characterize transcripts abundantly expressed in the human pituitary gland in general as well as to isolate novel transcripts expressed specifically in this gland, we generated an expression profile of the active genes transcribed in it. A total of 1015 randomly collected 3prime prime or minute expressed sequence tags (ESTs) (gene signatures, GSs) were grouped into 527GS species. The results showed the relative expression levels of genes in the pituitary gland. The genes comprising more than 1% of total mRNA were prolactin, growth hormone and chromogranin B genes. When known genes were categorized, the genes for pituitary hormones were the most actively transcribed, followed by the genes for ribosomal proteins, nuclear proteins and secretory granule proteins. Through comparison of this gene expression profile with the BodyMap database containing profiles generated from 63 other human tissues, we obtained 11 genes which appeared to be specifically expressed in the pituitary gland. In addition to the eight known genes, we identified three novel pituitary-specific transcripts which encode putative proteins: pituitary gland specific factor 1a (PGSF1a), PGSF1b and PGSF2. This expression profile method is a novel approach to the isolation of pituitary-specific genes that may have important functions.

Amino Acid Sequence↗