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cDNA Microarray (cdna + microarray)
Kinds of cDNA Microarray Terms modified by cDNA Microarray Selected AbstractsDifferentially expressed genes associated with CIS -diamminedichloroplatinum (II) resistance in head and neck cancer using differential display and CDNA microarrayHEAD & NECK: JOURNAL FOR THE SCIENCES & SPECIALTIES OF THE HEAD AND NECK, Issue 3 2003Eisaku Higuchi MD Abstract Background. The mechanism by which cancer cells become resistant to cis -Diamminedichloroplatinum (II) (cDDP) is not completely understood. To investigate the molecular markers involved in the cDDP resistance, we compared the gene expression profiles between a head and neck squamous cell carcinoma (HNSCC) line sensitive to cDDP and its cDDP-resistant variant. Methods. Both a fluorescent differential display and a cDNA microarray analysis were applied to distinguish the gene profiles between KB, a human HNSCC line, and its cDDP-resistant variant (KB/cDDP). These results were confirmed by Northern blot analysis. Results. One up-regulated gene, glycoprotein hormone ,-subunit, and two down-regulated genes coding membrane proteins, human folate receptor and tumor-associated antigen L6, were identified in KB/cDDP cells. Conclusions. Our findings suggest that development of the cDDP-resistant phenotype is accompanied by alternations of gene expression including a glycoprotein hormone and membrane proteins. These gene products could be new molecular markers for resistance to cDDP. © 2003 Wiley Periodicals, Inc. Head Neck 25: 187,193, 2003 [source] Transcriptional Analysis of Buffalo (Bubalus bubalis) Oocytes During In Vitro Maturation Using Bovine cDNA MicroarrayREPRODUCTION IN DOMESTIC ANIMALS, Issue 1 2010OM Kandil Contents The need for improving in vitro production of buffalo embryos necessitates a better understanding of the molecular mechanisms regulating early development including oocyte maturation. Here, we used bovine cDNA microarray platform to investigate mRNA abundance of buffalo oocytes before and after in vitro maturation. For this, a total of six pools each contains 50 immature or in vitro matured buffalo oocytes were used for mRNA isolation and subsequent cDNA synthesis. The BlueChip bovine cDNA microarray (with approximately 2000 clones) was used to analyse gene expression profiles between immature and matured oocytes. Statistical analysis of microarray data revealed a total of 104 transcripts to be differentially expressed between the two oocyte groups. Among these, transcription factors (ZFP91), M-phase mitotic cell cycle (MPHOSPH9), growth factor (BMP15) and DNA binding (HMGN2) were found to be up-regulated in immature oocytes. Similarly, matured oocytes were found to be enriched with genes involved in cytoskeleton (ACTB), hydrogen ion transporting (ATP6V1C2) and structural constituent of ribosome (RPS27A). Quantitative real-time polymerase chain reaction validated the expression profile of some selected transcripts during array analysis. In conclusion, to our knowledge, this is the first large-scale expression study to identify candidate genes differentially abundant and with potential role during buffalo oocyte maturation. [source] Developmental toxicity of estrogenic chemicals on rodents and other speciesCONGENITAL ANOMALIES, Issue 2 2002Taisen Iguchi ABSTRACT, Antenatal sex-hormone exposure induces lesions in mouse reproductive organs, which are similar to those in humans exposed in utero to a synthetic estrogen, diethylstilbestrol. The developing organisms including rodents, fish and amphibians are particularly sensitive to exposure to estrogenic chemicals during a critical window. Exposure to estrogens during the critical period induces long-term changes in reproductive as well as non-reproductive organs, including persistent molecular alterations. The antenatal mouse model can be utilized as an indicator of possible long-term consequences of exposure to exogenous estrogenic compounds including possible environmental endocrine disrupters. Many chemicals released into the environment potentially disrupt the endocrine system in wildlife and humans, some of which exhibit estrogenic activity by binding to the estrogen receptors. Estrogen responsive genes, therefore, need to be identified to understand the molecular basis of estrogenic actions. In order to understand molecular mechanisms of estrogenic chemicals on developing organisms, we are identifying estrogen responsive genes using cDNA microarray, quantitative RT-PCR, and differential display methods, and genes related to the estrogen-independent vaginal changes in mice induced by estrogens during the critical window. In this review, discussion of our own findings related to endocrine distuptor issue will be provided. [source] Dynamics of 17,-Ethynylestradiol exposure in rainbow trout (Oncorhynchus mykiss): Absorption, tissue distribution, and hepatic gene expression patternENVIRONMENTAL TOXICOLOGY & CHEMISTRY, Issue 11 2006Ann D. Skillman Abstract 17,-Ethynylestradiol (EE2) is a synthetic estrogen identified in sewage effluents. To understand better the absorption kinetics of EE2 and the induction of vitellogenin (VTG) and estrogen receptor , (ER,) mRNA, we subjected male rainbow trout (Onchorynchus mykiss) to continuous water exposures of 125 ng/L of EE2 for up to 61 d. Trout were either repetitively sampled for blood plasma or serially killed at selected time intervals. Vitellogenin, ER, mRNA, and EE2 were measured using enzymelinked immunosorbent assay and using quantitative polymerase chain reaction and gas chromatography,mass spectrometry, respectively. In separate experiments, trout were exposed to EE2 for 7 d, and hepatic gene expression was assessed using a low- and high-density cDNA microarray. The EE2 was rapidly absorbed by the trout, with an apparent equilibrium at 16 h in plasma and liver. The ER, mRNA levels also increased rapidly, reaching near-peak levels by 48 h. In contrast, plasma levels of VTG continuously increased for 19 d. After 61 d, tissues with the highest levels of VTG were the liver, kidney, and testes. Microarray-based gene expression studies provided unexpected results. In some cases, known estrogen-responsive genes (e.g., ER,) were unresponsive, whereas many of the genes that have no apparent link to estrogen function or EE2 toxicity were significantly altered in expression. Of the two microarray approaches tested in the present study, the high-density array appeared to be superior because of the improved quality of the hybridization signal and the robustness of the response in terms of the number of genes identified as being EE2 responsive. [source] Hepatitis C virus core protein activates ERK and p38 MAPK in cooperation with ethanol in transgenic miceHEPATOLOGY, Issue 4 2003Takeya Tsutsumi In human chronic hepatitis C, alcohol intake is a synergistic factor for the acceleration of hepatocarcinogenesis. Recently, we showed a significant increase of reactive oxygen species (ROS) in hepatitis C virus (HCV) core-transgenic mice fed ethanol-containing diets. Because previous studies indicated that ROS is closely associated with mitogen-activated protein kinases (MAPK), we examined activities of c-Jun N-terminal kinase, p38 MAPK, and extracellular signal-regulated kinase (ERK) in the liver of core-transgenic and nontransgenic mice with short-term ethanol feeding. Activity of ERK and p38 MAPK was increased in core-transgenic mice compared with nontransgenic mice, whereas neither ERK nor p38 MAPK was activated in core-transgenic mice with normal diets. In addition, activity of cyclic-AMP and serum responsive element, downstream pathways of p38 MAPK and ERK, was also increased. Comparison of gene expression profiles by cDNA microarray and real-time PCR revealed that galectin-1, which is associated with cell transformation, was significantly increased in ethanol-fed core-transgenic mice. On the other hand, glutathione S-transferase (GST), which plays a key role in protecting cells from oxidative stress, was decreased. In conclusion, these results suggest that HCV core protein cooperates with ethanol for the activation of some MAPK pathways, and leads to the modulation of several genes, contributing to the pathogenesis of liver disease of HCV- infected patients with high ethanol consumption. (Hepatology 2003;38:820,828). [source] Molecular characterization of the response to chemotherapy in conventional osteosarcomas: Predictive value of HSD17B10 and IFITM2INTERNATIONAL JOURNAL OF CANCER, Issue 4 2009Sébastien Salas Abstract The therapy regimen of high-grade osteosarcoma includes chemotherapy followed by surgical resection and postoperative chemotherapy. The degree of necrosis following definitive surgery remains the only reliable prognostic factor and is used to guide the choice of postoperative chemotherapy. The aim of this study was to find molecular markers able to classify patients with an osteosarcoma as good or poor responders to chemotherapy before beginning treatment. Gene expression screening of 20 nonmetastatic high-grade osteosarcoma patients was performed using cDNA microarray. Expression of selected relevant genes was validated using QRT-PCR. Immunohistochemistry on tissue microarrays sections of 73 biopsies was performed to investigate protein expression. Fluorescent in situ hybridization was performed for RPL8 gene. We have found that HSD17B10 gene expression was up-regulated in poor responders and that immunohistochemistry expression of HSD17B10 on biopsy before treatment was correlatedto response to chemotherapy. Other results include correlationof IFITM2, IFITM3, and RPL8 gene expression to chemotherapy response. A statistical correlation was found between polysomy 8 or gain of RPL8 and good response to chemotherapy. These data suggest that HSD17B10, RPL8, IFITM2, and IFITM3 genes are involved in the response to the chemotherapy and that HSD17B10 may be a therapeutic target. RPL8 and IFITM2 may be useful in the assessment at diagnosis and for stratifying patients taking part in randomized trials. © 2009 UICC [source] Whole genome analysis for liver metastasis gene signatures in colorectal cancerINTERNATIONAL JOURNAL OF CANCER, Issue 9 2007Dong Hyuk Ki Abstract Liver metastasis is one of the major causes of death in colorectal cancer (CRC) patients. To understand this process, we investigated whether the gene expression profiling of matched colorectal carcinomas and liver metastases could reveal key molecular events involved in tumor progression and metastasis. We performed experiments using a cDNA microarray containing 17,104 genes with the following tissue samples: paired tissues of 25 normal colorectal mucosa, 27 primary colorectal tumors, 13 normal liver and 27 liver metastasis, and 20 primary colorectal tumors without liver metastasis. To remove the effect of normal cell contamination, we selected 4,583 organ-specific genes with a false discovery rate (FDR) of 0.0067% by comparing normal colon and liver tissues using significant analysis of microarray, and these genes were excluded from further analysis. We then identified and validated 46 liver metastasis-specific genes with an accuracy of 83.3% by comparing the expression of paired primary colorectal tumors and liver metastases using prediction analysis of microarray. The 46 selected genes contained several known oncogenes and 2 ESTs. To confirm that the results correlated with the microarray expression patterns, we performed RT-PCR with WNT5A and carbonic anhydrase II. Additionally, we observed that 21 of the 46 genes were differentially expressed (FDR = 2.27%) in primary tumors with synchronous liver metastasis compared with primary tumors without liver metastasis. We scanned the human genome using a cDNA microarray and identified 46 genes that may play an important role in the progression of liver metastasis in CRC. © 2007 Wiley-Liss, Inc. [source] Predicting 5-fluorouracil chemosensitivity of liver metastases from colorectal cancer using primary tumor specimens: Three-gene expression model predicts clinical responseINTERNATIONAL JOURNAL OF CANCER, Issue 2 2006Ryusei Matsuyama Abstract We identified genes related to 5-fluorouracil (5-FU) sensitivity in colorectal cancer and utilized these genes for predicting the 5-FU sensitivity of liver metastases. Eighty-one candidate genes involved in 5-FU resistance in gastric and colon cancer cell lines were previously identified using a cDNA microarray. In this study, the mRNA expression levels of these 81 selected genes and the genes of 5-FU-related enzymes, including thymidylate synthase (TS), dihydropyrimidine dehydrogenase (DPD) and orotate phosphoribosyltransferase (OPRT), were measured using real-time quantitative RT-PCR assays of surgically resected materials from primary colorectal tumors in 22 patients. Clinical responses were estimated by evaluating the effects of 5-FU-based hepatic artery injection (HAI) chemotherapy for synchronous liver metastases. Four genes (TNFRSF1B, SLC35F5, NAG-1 and OPRT) had significantly different expression profiles in 5-FU-nonresponding and responding tumors (p < 0.05). A "Response Index" system using three genes (TNFRSF1B, SLC35F5 and OPRT) was then developed using a discriminate analysis; the results were well correlated with the individual chemosensitivities. Among the 11 cases with positive scores in our response index, 9 achieved a reduction in their liver metastases after 5-FU-based chemotherapy, whereas only 1 of the 11 cases with negative scores responded well to chemotherapy. Our "Response Index" system, consisting of TNFRSF1B, SLC35F5 and OPRT, has great potential for predicting the efficacy of 5-FU-based chemotherapy against liver metastases from colorectal cancer. © 2006 Wiley-Liss, Inc. [source] Analysis of gene expression profiles in human HL-60 cell exposed to cantharidin using cDNA microarrayINTERNATIONAL JOURNAL OF CANCER, Issue 2 2004Jun-Ping Zhang Abstract Cantharidin is a natural toxin that has antitumor properties and causes leukocytosis as well as increasing sensitivity of tumor cells resistant to other chemotherapeutic agents. There is limited information, however, on the molecular pharmacological mechanisms of cantharidin on human cancer cells. We have used cDNA microarrays to identify gene expression changes in HL-60 promyeloid leukemia cells exposed to cantharidin. Cantharidin-treated cells not only decreased expression of genes coding for proteins involved in DNA replication (e.g., DNA polymerase delta), DNA repair (e.g., FANCG, ERCC), energy metabolism (e.g., isocitrate dehydrogenase alpha, ADP/ATP translocase), but also decreased expression of genes coding for proteins that have oncogenic activity (e.g., c-myc, GTPase) or show tumor-specific expression (e.g., phosphatidylinositol 3-kinase). In contrast, these treated cells overexpressed several genes that encode intracellular and secreted growth-inhibitory proteins (e.g., BTG2, MCP-3) as well as proapoptotic genes (e.g., ATL-derived PMA-responsive peptide). Our findings suggest that alterations in specific genes functionally related to cell proliferation or apoptosis may be responsible for cantharidin-mediated cytotoxicity. We also found that exposure of HL-60 cells to cantharidin resulted in the decreased expression of multidrug resistance-associated protein genes (e.g., ABCA3, MOAT-B), suggesting that cantharidin may be used as an oncotherapy sensitizer, and the increased expression of genes in modulating cytokine production and inflammatory response (e.g., NFIL-3, N-formylpeptide receptor), which may partly explain the stimulating effects on leukocytosis. Our data provide new insight into the molecular mechanisms of cantharidin. © 2003 Wiley-Liss, Inc. [source] Identification of Novel Target Genes of the Bone-Specific Transcription Factor Runx2,JOURNAL OF BONE AND MINERAL RESEARCH, Issue 6 2004Michael Stock Abstract Fifteen putative transcriptional target genes regulated by the osteogenic transcription factor Runx2 were identified by cDNA microarray and differential hybridization techniques. Expression pattern and regulation of one gene, Pttg1ip, was analyzed in detail. Introduction: The transcription factor Runx2 is a key regulator of osteoblast development and plays a role in chondrocyte maturation. The identification of transcriptional target genes of Runx2 may yield insight into how osteoblastic differentiation is achieved on a molecular level. Materials and Methods: Using a differential hybridization technique (selective amplification through biotin and restriction-mediated enrichment [SABRE]) and cDNA microarray analysis, 15 differentially expressed genes were identified using mRNA from C3H 10T1/2 cells with constitutive and inducible overexpression of Runx2. Results and Conclusions: Among the 15 genes identified, 4 encode the extracellular matrix proteins Ecm1, Mgp, Fbn5, and Osf-2, three represent the transcription factors Esx1, Osr1, and Sox9, whereas others were Ptn, Npdc-1, Hig1, and Tem1. The gene for Pttg1ip was upregulated in Runx2-expressing cells. Pttg1ip is widely expressed during development, but at highest levels in limbs and gonads. The Pttg1ip promoter binds Runx2 in a sequence specific manner, and Runx2 is able to transactivate the Pttg1ip promoter in MC3T3-E1 cells. Therefore, Pttg1ip is likely to be a novel direct transcriptional target gene of Runx2. In conclusion, the genes identified in this study are important candidates for mediating Runx2 induced cellular differentiation. [source] Recombinant vascular basement membrane derived multifunctional peptide blocks endothelial cell angiogenesis and neovascularization,JOURNAL OF CELLULAR BIOCHEMISTRY, Issue 2 2010Chengkun Wang Abstract Angiogenesis is an innovative target in the therapy of cancer and other diseases, but the effects of anti-angiogenic drugs have been rather modest in clinical trials. We have developed a small peptide, recombinant vascular basement membrane derived multifunctional peptide (rVBMDMP), which significantly inhibits endothelial cells in vitro. Here we test the mechanisms of rVBMDMP in angiogenesis balance in assays of tubule formation, colony formation, and apoptosis in HUVE-12 endothelial cells. We also analyzed the differential expression of phosphorylation proteins and related genes in a protein phosphorylation chip and extracellular matrix adhesion molecule cDNA microarray, and validated changes with Western blot or real-time quantitative PCR, respectively. rVBMDMP dose-dependently inhibited colony formation, induced apoptosis, and inhibited in vitro tubule formation. rVBMDMP increased the phosphorylation of 88 signal proteins, including caspase-3, death receptor 3, 4, and 5, and integrin ,V, ,1, and ,3, and down-regulated 41 signal proteins, including EGFR, pEGFR, VEGFR-1, and survivin versus control. rVBMDMP upregulated 14 genes, including collagen 4, 7, and 27, and down-regulated 21 genes, including integrin ,V,3, MMP10, and MMP12. Our study suggests that rVBMDMP inhibits angiogenesis and may be a viable drug candidate in anti-angiogenesis and anticancer therapies. J. Cell. Biochem. 111: 453,460, 2010. © 2010 Wiley-Liss, Inc. [source] DNA methylation and histone modification regulate silencing of OPG during tumor progression,JOURNAL OF CELLULAR BIOCHEMISTRY, Issue 1 2009Tung-Ying Lu Abstract The identification of molecules that are down-regulated in malignant phenotype is important for understanding tumor biology and their role in tumor suppression. We compared the expression profile of four normal nasal mucosal (NNM) epithelia and a series of nasopharyngeal cancinoma (NPC) cell lines using cDNA microarray and confirmed the actual expression of the selected genes, and found osteoprotegerin (OPG) to be ubiquitously deficient in NPC cells. We also found OPG to be down-regulated in various cancer cell lines, including oral, cervical, ovarian, lung, breast, pancreas, colon, renal, prostate cancer, and hepatoma. Administration of recombinant OPG (rOPG) brought about a reduction in cancer cell growth through apoptotic mechanism. We generated eleven monoclonal antibodies (MAbs) against OPG to study OPG's expression and biological functions in cancer cells. OPG was detected in the tumor stromal regions, but not in the cancer cell per se in surgical specimens of liver cancer. Quantitative reverse transcription-polymerase chain reaction (Q-RT-PCR) revealed that OPG was down-regulated in NPC tissues compared with normal nasal polyp (NNP) tissues. In addition, we showed OPG silencing to be associated with promoter methylation as well as histone modifications. In OPG-silenced cancer cell lines, the OPG gene promoter CpG dinucleotides were highly methylated. Compared to normal cells, silenced OPG gene in cancer cells were found to have reduced histone 3 lysine 4 tri-methylation (H3K4me3) and increased histone 3 lysine 27 tri-methylation (H3K27me3). Taken together, these results suggest that OPG silencing in carcinoma cancer cells occurs through epigenetic repression. J. Cell. Biochem. 108: 315,325, 2009. © 2009 Wiley-Liss, Inc. [source] Expansion of the genomics research on Atlantic salmon Salmo salar L. project (GRASP) microarray toolsJOURNAL OF FISH BIOLOGY, Issue 9 2008K. R. VonSchalburg Salmonids are the most widely studied group of fish, and in the last few years, genomics technologies have begun to contribute to this rich biology. The first salmonid microarrays appeared in 2004 and since then several dozen studies have demonstrated the utility of genomic approaches. The widespread use of the genomics research on Atlantic salmon project 16 k array and greatly expanded genome resources have led to the development of an experimental 5 k oligo (70-mer) array and a 32 k cDNA microarray in the near future. In this paper, the authors examined some of the procedures used in the development of past arrays and reexamined them in light of new genomic data available. Some preliminary control experiments of the new 5 k array were investigated that examine oligo designs based on distance from the polyA tail, the effects of mismatches and cross-species hybridization specificity. Beneficial approaches are also identified in the development of the new 32 k cDNA array. [source] c-DNA Microarray to determine molecular events in neurodegeneration and neuroprotectionJOURNAL OF NEUROCHEMISTRY, Issue 2002M. B. H. Youdim Cell death in CNS involves complex processes, many of which have not been identified biochemically. At the present biochemical techniques cannot adequately establish these. However, the advent of cDNA microarray or microchips, in which the expression of thousands of genes can be measured at once to give a global assessment in disease pathology, its progress or animal models, has simplified this. We have employed this technique to study the mechanism of neurotoxicity of MPTP and 6-hydroxydoapmine induced in neuronally derived cells in culture, in the animal models of Parkinson's disease and neuroprotection initiated by monoamine oxidase B inhibitor, rasagiline; iron chelators, R-apomorphine and EGCG and other neuroprotective drugs. Our studies have clearly indicated that MPTP induced early gene expression, prior to cell death (first 24 h), are prerequirement for 51 late gene expression changes implicated at the time of neuronal death. The latter genes include those involved in iron metabolism, oxidative stress, inflammatory processes, glutaminergic excitotoxicity, nitric oxide, growth factors, transcription factors, cell cycle, intermediatory metabolism and other gene previously not identified. The expressions of many of the latter genes, also identified by in situ hybridization, are prevented when the animals are pretreated with the above neuroprotective drugs. These studies have clearly shown that neurodegeneratrion is a complex cascades of ,domino' effect. Thus a single neuroprotective drug treatment may not be adequate to prevent it, but, that a cocktail of drugs might. [source] The Th1/Th2 immune-type response of the recurrent aphthous ulceration analyzed by cDNA microarrayJOURNAL OF ORAL PATHOLOGY & MEDICINE, Issue 3 2004R. C. Borra Background:, The reduced ability to activate oral tolerance plays a role in the pathogenesis of some gastrointestinal inflammatory diseases. This activation may reflect a preferential reduction of a T-helper (Th)2- or Th3-type response. In recurrent aphthous ulceration (RAU), genetic and environmental factors may contribute to low tolerance, permitting a cytotoxic reaction against the oral epithelium. The cytokine profile has not permitted the definition of RAU as resulting from enhanced Th1 or Th2 responses. A cDNA microarray study would allow the identification of differentially expressed genes and provide a basis for classification of the immune response. Methods:, The cDNA from 29 samples of aphthae and from 11 samples of normal mucosa from aphthae-free volunteers were hybridized on microarray membranes with 1176 genes. Results:, Forty-one differentially expressed genes were identified, and a higher expression level of the Th1 gene cluster in RAU was found. Conclusions:, Microarrays permitted us definition of the gene expression profile of the lesion and identify an increased Th1 activity in RAU lesions. [source] Paclitaxel induces apoptosis via caspase-3 activation in human osteogenic sarcoma cells (U-2 OS)JOURNAL OF ORTHOPAEDIC RESEARCH, Issue 5 2005K.-H. Lu Abstract Paclitaxel has been found to exhibit cytotoxic and antitumor activity. There is little information regarding the mechanisms of apoptotic-inducing effect of paclitaxel on human osteogenic sarcoma U-2 OS cells. Several key regulatory proteins are involved in the initiation of apoptosis. Caspase-3 plays a direct role in proteolytic cleavage of cellular proteins responsible for progression to apoptosis. We examined the effect of paclitaxel on the cell cycle arrest and apoptosis in U-2 OS cells using flow cytometric analysis and Western blotting. We also measured the inhibition of paclitaxel-induced apoptosis and the caspase-3 activity by the broad-spectrum caspase inhibitor z-VAD-fmk on U-2 OS cells. The increased levels of casapse-3 were also confirmed by cDNA microarray. Our observations were: (1) paclitaxel treatment resulted in G2/M-cycle arrest in U-2 OS cells; (2) time and dose dependent apoptosis of U-2 OS cells was induced by paclitaxel; (3) in U-2 OS cells, z-VAD-fmk blocked the paclitaxel-induced apoptosis and caspase-3 activation. These results suggest that paclitaxel-induced G2/M-cycle arrest of the G2/M phase and apoptosis via a caspase-3 pathway in U-2 OS cells. © 2005 Orthopaedic Research Society. Published by Elsevier Ltd. All rights reserved. [source] The knockdown of endogenous replication factor C4 decreases the growth and enhances the chemosensitivity of hepatocellular carcinoma cellsLIVER INTERNATIONAL, Issue 1 2009Masaaki Arai Abstract Aims: To identify differentially expressed genes and thereby detect potential molecular targets for future therapies directed against hepatocellular carcinoma (HCC). Methods: To isolate differentially expressed genes between HCC and adjacent non-cancerous liver tissues, cDNA microarray and quantitative reverse transcriptase polymerase chain reaction analyses were performed. Gene knockdown experiments in HepG2 cells were also performed using small interfering RNAs (siRNAs). Proteins were detected by immunostaining, and cell proliferation was analysed using the MTT/WST-8 assay. Apoptosis and cell cycle analyses were performed using flow cytometry. Results: After an intensive screening for differentially expressed genes in HCC tissues, we isolated 23 upregulated genes in these lesions. Among these, we focused on the replication factor C4 (RFC4) gene. The expression of endogenous RFC4 proteins in HepG2 cells was found to be significantly reduced by RFC4 -specific siRNA. This inhibition of RFC4 expression correlated with a decrease in cellular proliferation, increased levels of apoptosis and a sensitizing of the cells to the DNA-damaging chemotherapeutic agents, doxorubicin and camptothecin. Conclusion: The replication factor C4 gene may be a novel target for developing cancer therapeutics, which can enhance the antitumour activity of chemotherapeutic agents that induce DNA damage. [source] The transcriptomics of life-history trade-offs in whitefish species pairs (Coregonus sp.)MOLECULAR ECOLOGY, Issue 7 2008J. ST-CYR Abstract Despite the progress achieved in elucidating the ecological mechanisms of adaptive radiation, there has been little focus on documenting the extent of adaptive differentiation in physiological functions during this process. Moreover, a thorough understanding of the genomic basis underlying phenotypic adaptive divergence is still in its infancy. One important evolutionary process for which causal genetic mechanisms are largely unknown pertains to life-history trade-offs. We analysed patterns of gene transcription in liver tissue of sympatric dwarf and normal whitefish from two natural lakes, as well as from populations reared in controlled environments, using a 16 006-gene cDNA microarray in order to: (i) document the extent of physiological adaptive divergence between sympatric dwarf and normal species pairs, and (ii) explore the molecular mechanisms of differential life history trade-offs between growth and survival potentially involved in their adaptive divergence. In the two natural lakes, 6.45% of significantly transcribed genes showed regulation either in parallel fashion (2.39%) or in different directions (4.06%). Among genes showing parallelism in regulation patterns, we observed a higher proportion of over-expressed genes in dwarf relative to normal whitefish (70.6%). Patterns observed in controlled conditions were also generally congruent with those observed in natural populations. Dwarf whitefish consistently showed significant over-expression of genes potentially associated with survival through enhanced activity (energy metabolism, iron homeostasis, lipid metabolism, detoxification), whereas more genes associated with growth (protein synthesis, cell cycle, cell growth) were generally down-regulated in dwarf relative to normal whitefish. Overall, parallelism in patterns of gene transcription, as well as patterns of interindividual variation across controlled and natural environments, provide strong indirect evidence for the role of selection in the evolution of differential regulation of genes involving a vast array of potentially adaptive physiological processes between dwarf and normal whitefish. Our results also provide a first mechanistic, genomic basis for the observed trade-off in life-history traits distinguishing dwarf and normal whitefish species pairs, wherein enhanced survival via more active swimming, necessary for increased foraging and predator avoidance, engages energetic costs that translate into slower growth rate and reduced fecundity in dwarf relative to normal whitefish. [source] CDNA microarray analysis of gene expression in fibroblasts of patients with x-linked Emery,Dreifuss muscular dystrophyMUSCLE AND NERVE, Issue 6 2002Toshifumi Tsukahara PhD Abstract To clarify the molecular nature of the pathogenesis in X-linked Emery,Dreifuss muscular dystrophy (EDMD), we monitored the expression of 2400 genes in control and EDMD fibroblasts by using complementary DNA (cDNA) microarray techniques. A total of 60 genes whose expression was altered in EDMD fibroblasts when compared with control fibroblasts were identified. Twenty-eight genes whose expression was altered with the emerin deficiency were rescued by infection with a recombinant adenovirus expressing emerin. The altered expression in five genes, including the lamin A/C gene, was confirmed by reverse transcription,polymerase chain reaction. Our preliminary results suggest a correlation between disease similarity and gene expression. We conclude that the cDNA microarray is a very efficient tool to clarify genetic and pathological features of diseases. © 2002 Wiley Periodicals, Inc. Muscle Nerve 25: 898,901, 2002 [source] Gene expression profiling of Dunaliella sp. acclimated to different salinitiesPHYCOLOGICAL RESEARCH, Issue 1 2010Minjung Kim SUMMARY To investigate which genes may be important for growth under extreme conditions such as very low or high salinities, a survey of the Dunaliella sp. transcriptome was performed with a cDNA microarray which had been generated previously representing 778 expressed sequence tags. The comparative microarray analysis indicated that 142 genes differed in expression levels by more than twofold in cells grown at extreme salinities (0.08 M and 4.5 M NaCl) when compared with cells grown at intermediate salinity (1.5 M NaCl). Of these genes, 28 had increased expression and 57 were suppressed in cells grown at low salinity. In cells grown at high salinity, 43 genes showed increased expression and 69 genes showed suppressed expression. However, we did observe a large overlap in the expression of extreme salinity-responsive genes based on Venn diagram analysis, which found 55 genes that responded to both of the two extreme salinity conditions. Further, we found that several genes had similar expression levels under low and high salinities, including some general stress response genes that were upregulated in both extreme salinity conditions. For confirmation of the validity of the cDNA microarray analysis, expression of several genes was independently confirmed by the use of gene-specific primers and real-time polymerase chain reaction. The present study is the first large-scale comparative survey of the transcriptome from the microalga Dunaliella sp. acclimated to extreme salinities, thus providing a platform for further functional investigation of differentially expressed genes in Dunaliella. [source] Evaluation of a Non-Targeted "Omic" Approach in the Safety Assessment of Genetically Modified PlantsPLANT BIOLOGY, Issue 5 2006S. B. Metzdorff Abstract: Genetically modified plants must be approved before release in the European Union, and the approval is generally based upon a comparison of various characteristics between the transgenic plant and a conventional counterpart. As a case study, focusing on safety assessment of genetically modified plants, we here report the development and characterisation of six independently transformed Arabidopsis thaliana lines modified in the flavonoid biosynthesis. Analyses of integration events and comparative analysis for characterisation of the intended effects were performed by PCR, quantitative Real-time PCR, and High Performance Liquid Chromatography. Analysis by cDNA microarray was used as a non-targeted approach for the identification of potential unintended effects caused by the transformation. The results revealed that, although the transgenic lines possessed different types of integration events, no unintended effects were identified. However, we found that the majority of genes showing differential expression were identified as stress-related genes and that environmental conditions had a large impact on the expression of several genes, proteins, and metabolites. We suggest that the microarray approach has the potential to become a useful tool for screening of unintended effects, but state that it is crucial to have substantial information on the natural variation in traditional crops in order to be able to interpret "omics" data correctly within the framework of food safety assessment strategies of novel plant varieties, including genetically modified plant varieties. [source] Protein chip-based microarray profiling of oxidized low density lipoprotein-treated cellsPROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 5 2005Sergiy Sukhanov Abstract Commercially available high-content Ab380 and extensively validated DLM26 homemade protein microarrays were used to profile the effects of the pro-atherogenic molecule, oxidized low density lipoprotein (OxLDL), on human aortic smooth muscle cells. Protein microarrays detected 298 proteins in cell lysates and 54 of these were differentially regulated. Microarray data were validated by immunoblotting for a selected set of up- and down-regulated proteins. The protein microarray data sets were compared with our recent cDNA microarray-based gene expression results in order to characterize the global effect of OxLDL on smooth muscle cell functions. A group of cell-cell interaction molecules was classified as up-regulated by OxLDL, whereas nucleic acid/protein biosynthesis, structural and humoral response proteins/genes were under-expressed in cells treated by OxLDL. These findings reveal the major pattern of OxLDL-induced effects on the human aortic smooth muscle cells functions and also demonstrate that protein chip-based microarrays could be a useful proteomic tool to profile disease-related states of muscle cells. [source] Freezing-sensitive tomato has a functional CBF cold response pathway, but a CBF regulon that differs from that of freezing-tolerant ArabidopsisTHE PLANT JOURNAL, Issue 6 2004Xin Zhang Summary Many plants increase in freezing tolerance in response to low temperature, a process known as cold acclimation. In Arabidopsis, cold acclimation involves action of the CBF cold response pathway. Key components of the pathway include rapid cold-induced expression of three homologous genes encoding transcriptional activators, CBF1, 2 and 3 (also known as DREB1b, c and a, respectively), followed by expression of CBF-targeted genes, the CBF regulon, that increase freezing tolerance. Unlike Arabidopsis, tomato cannot cold acclimate raising the question of whether it has a functional CBF cold response pathway. Here we show that tomato, like Arabidopsis, encodes three CBF homologs, LeCBF1,3 (Lycopersicon esculentum CBF1,3), that are present in tandem array in the genome. Only the tomato LeCBF1 gene, however, was found to be cold-inducible. As is the case for Arabidopsis CBF1,3, transcripts for LeCBF1,3 did accumulate in response to mechanical agitation, but not in response to drought, ABA or high salinity. Constitutive overexpression of LeCBF1 in transgenic Arabidopsis plants induced expression of CBF-targeted genes and increased freezing tolerance indicating that LeCBF1 encodes a functional homolog of the Arabidopsis CBF1,3 proteins. However, constitutive overexpression of either LeCBF1 or AtCBF3 in transgenic tomato plants did not increase freezing tolerance. Gene expression studies, including the use of a cDNA microarray representing approximately 8000 tomato genes, identified only four genes that were induced 2.5-fold or more in the LeCBF1 or AtCBF3 overexpressing plants, three of which were putative members of the tomato CBF regulon as they were also upregulated in response to low temperature. Additional experiments indicated that of eight tomato genes that were likely orthologs of Arabidopsis CBF regulon genes, none were responsive to CBF overexpression in tomato. From these results, we conclude that tomato has a complete CBF cold response pathway, but that the tomato CBF regulon differs from that of Arabidopsis and appears to be considerably smaller and less diverse in function. [source] Identification of cold-inducible downstream genes of the Arabidopsis DREB1A/CBF3 transcriptional factor using two microarray systemsTHE PLANT JOURNAL, Issue 6 2004Kyonoshin Maruyama Summary The transcriptional factor DREB/CBF (dehydration-responsive element/C-repeat-binding) specifically interacts with the dehydration-responsive element (DRE)/C-repeat (CRT) cis -acting element (A/GCCGAC) and controls the expression of many stress-inducible genes in Arabidopsis. Transgenic plants overexpressing DREB1A showed activated expression of many stress-inducible genes and improved tolerance to not only drought, salinity, and freezing but also growth retardation. We searched for downstream genes in transgenic plants overexpressing DREB1A using the full-length cDNA microarray and Affymetrix GeneChip array. We confirmed candidate genes selected by array analyses using RNA gel blot and identified 38 genes as the DREB1A downstream genes, including 20 unreported new downstream genes. Many of the products of these genes were proteins known to function against stress and were probably responsible for the stress tolerance of the transgenic plants. The downstream genes also included genes for protein factors involved in further regulation of signal transduction and gene expression in response to stress. The identified genes were classified into direct downstream genes of DREB1A and the others based on their expression patterns in response to cold stress. We also searched for conserved sequences in the promoter regions of the direct downstream genes and found A/GCCGACNT in their promoter regions from ,51 to ,450 as a consensus DRE. The recombinant DREB1A protein bound to A/GCCGACNT more efficiently than to A/GCCGACNA/G/C. [source] Simultaneously detection of genomic and expression alterations in prostate cancer using cDNA microarray,THE PROSTATE, Issue 14 2008Mei Jiang Abstract BACKGROUND Prostate cancer is a common disease among men but the knowledge of the prostate carcinogenesis is still limited. METHODS cDNA microarray-based comparative genomic hybridization (CGH) and expression profiling were performed to screen the genomic and the expression changes in prostate cancer respectively. The two data were integrated to study the influence of genomic aberrations on gene expression and seek for the genes with their expression affected by the genomic aberrations. Real-time PCR was performed to evaluate the array data. RESULTS Array-based CGH detected gains at 2q, 3p/q, 5q, 6q, 8q, 9p, 10p/q, 11q, 12p, 14q, and 19p/q and losses at 1p, 2p, 4q, 6p/q, 7p, 11p/q, 12q, 17p/q, 19p/q, and Xp/q in more than 20% prostate tumors and narrowed these aberrations. For example, the gain of 8q was mapped to five minimal regions. Novel aberrations were also identified, such as loss at Xq21.33-q22.2. Expression profiling discovered the significant biological processes involved in the prostate carcinogenesis, such as exogenous antigen presentation via MHC class II and protein ubiquitination. Integration analysis revealed a weak positive correlation between genomic copy number and gene expression level. Fifty-three genes showed their expression directly affected by the genomic aberrations possibly, including more than one member of Ras superfamily and major histocompatibility complex (MHC). These genes are involved in multiple biological processes. CONCLUSIONS Integration of the CGH and expression data provided more information than separate analysis. Although the direct influence of genomic aberrations on gene expression seems weak, the influence can be extended by indirect regulation through a few directly affected genes. Because the influence can be persistent, the genes directly affected by the genomic aberrations may play key roles in the prostate carcinogenesis and are worth further analysis. Prostate 68: 1496,1509, 2008. © 2008 Wiley-Liss, Inc. [source] Complex immunomodulatory effects of interferon-, in multiple sclerosis include the upregulation of T helper 1-associated marker genesANNALS OF NEUROLOGY, Issue 3 2001Klaus-Peter Wandinger MD Multiple sclerosis (MS) is considered an autoimmune disease that is mediated by proinflammatory T helper-1 lymphocytes. The putative mechanism of interferon-, (IFN-,), an approved treatment for MS, includes the inhibition of T-cell proliferation, blocking of blood-brain-barrier opening and T-cell transmigration into the brain via interference with cell adhesion, and the upregulation of anti-inflammatory (TH2) cytokines. In the present study, a gene expression analysis of IFN-,-treated peripheral blood mononuclear cells by cDNA microarray documents the broad effects of IFN-, that are not purely anti-inflammatory. Specifically, we addressed the effect of IFN-, on T helper-1 differentiation- or lineage markers such as the IL-12 receptor ,2 chain and the chemokine receptor CCR5 that have been implicated in the pathogenesis of MS. Both markers were significantly upregulated in vitro and in vivo under IFN-, therapy, supporting that this cytokine exerts complex effects on the immune system. The combination of cDNA microarray and quantitative polymerase chain reaction will expand our knowledge of the immunological effects of such pleiotropic agents as IFN-,, may provide a key to why certain patients fail to respond, and eventually influence our view of the disease pathogenesis. [source] Mining plant diversity: Gerbera as a model system for plant developmental and biosynthetic researchBIOESSAYS, Issue 7 2006Teemu H. Teeri Gerbera hybrida is a member of the large sunflower family (Asteraceae). Typical of Asteraceae, Gerbera bears different types of flowers in its inflorescence. The showy marginal flowers comprise elongate, ligulate corollas that are female, whereas the central and inconspicuous disc flowers are complete, with both male and female organs. As such, Gerbera offers great potential for comparative developmental research within a single genotype. Moreover, different Gerbera varieties show an impressive spectrum of color patterns, directly displaying responses to developmental cues at all important morphological levels (flower type, flower organ and within organs). Further, Gerbera harbors an arsenal of Asteraceae-type secondary metabolites, not present in other model plants. With powerful reverse genetics methods, a large collection of EST sequences and a new cDNA microarray, Gerbera has become a model plant of the sunflower family. BioEssays 28: 756,767, 2006. © 2006 Wiley Periodicals, Inc. [source] Involvement of kinesin family member 2C/mitotic centromere-associated kinesin overexpression in mammary carcinogenesisCANCER SCIENCE, Issue 1 2008Arata Shimo To elucidate the molecular mechanisms of mammary carcinogenesis and discover novel therapeutic targets for breast cancer, we previously carried out genome-wide expression profile analysis of 81 breast cancer cases by means of cDNA microarray coupled with laser microbeam microdissection of cancer cells. Among the dozens of transactivated genes, in the present study we focused on the functional significance of kinesin family member 2C (KIF2C)/mitotic centromere-associated kinesin (MCAK) in the growth of breast cancer cells. Northern blot and immunohistochemical analyses confirmed KIF2C/MCAK overexpression in breast cancer cells, and showed that it is expressed at undetectable levels in normal human tissues except the testis, suggesting KIF2C/MCAK to be a cancer,testis antigen. Western blot analysis using breast cancer cell lines revealed a significant increase in the endogenous KIF2C/MCAK protein level and its phosphorylation in G2/M phase. Treatment of breast cancer cells with small interfering RNA against KIF2C/MCAK effectively suppressed KIF2C/MCAK expression and inhibited the growth of the breast cancer cell lines T47D and HBC5. In addition, we found that KIF2C/MCAK expression was significantly suppressed by ectopic introduction of p53. These findings suggest that overexpression of KIF2C/MCAK might be involved in breast carcinogenesis and is a promising therapeutic target for breast cancers. (Cancer Sci 2008; 99: 62,70) [source] Simvastatin inactivates ,1-integrin and extracellular signal-related kinase signaling and inhibits cell proliferation in head and neck squamous cell carcinoma cellsCANCER SCIENCE, Issue 6 2007Ikuko Takeda The 3-hydroxy-3-methylglutaryl (HMG)-CoA reductase inhibitors, also called statins, are commonly used as lipid-lowering drugs that inhibit cholesterol biosynthesis. An anticancer effect, as a pleiotropic function of certain statins, has been hypothesized. In the present study, we investigated the effect of simvastatin, one of the natural statins, on cell proliferation, cell cycle, invasive activity, and molecular expressions associated with cell,extracellular matrix adhesion, signal transduction, and DNA synthesis in Tu167 and JMAR cells from head and neck squamous cell carcinoma. The addition of simvastatin resulted in a dose-dependent inhibition of cell growth and migration into the extracellular matrix. Considerable morphological changes occurred after treatment with simvastatin, demonstrating loss of cell adhesion and disruption of actin filaments in cytoplasm. The inhibitory effect of simvastatin on cell proliferation seemed to be associated with cell cycle arrest and increased expression of p21, p27, and activated caspase-3. The expression of ,1-integrin, a counter adhesion for the extracellular matrix, phosphorylated FAK, and phosphorylated ERK was decreased by treatment with simvastatin. The proapoptotic effect of simvastatin was inhibited by treatment with mevalonate. cDNA microarray assay demonstrated that molecular changes resulting from treatment with simvastatin included the up-regulation of cell cycle regulators and apoptosis-inducing factors and the down-regulation of integrin-associated molecules and cell proliferation markers. Of down-regulated genes induced by simvastatin treatment, a significant depletion of thymidylate synthase was confirmed using western blot analysis. These results imply that simvastatin has the potential to be effective for the prevention of the growth and metastasis of cancer cells. (Cancer Sci 2007; 98: 890,899) [source] Chemosensitivity of human pancreatic carcinoma cells is enhanced by IkB, super-repressorCANCER SCIENCE, Issue 5 2003Toshiyuki Sato Pancreatic cancer has an unfavorable prognosis; surgery and chemotherapy at present have only limited value. To improve the prognosis of pancreatic cancer, effective non-surgical therapy is necessary. NF- kB is reported to be related to resistance to apopto-sis, but its role in Chemosensitivity remains controversial. We examined the effects on Chemosensitivity of inhibition by induction of the super-repressor IkB, in pancreatic cancer cell lines, BxPC-3, Capan-1 and Panc-1. IkB, protein was transduced by infection of adenovirus vector AxCAhlkB,N. Sensitivity to VP-16 and doxorubicin was increased significantly by IkB, induction in all three pancreatic cell lines. To investigate molecular events during IkB, induction, we examined the changes in expression of drug-resistance-related genes by real-time RT-PCR and those in apoptosis-related genes by cDNA microarray. There was no common change of gene expression before and after IkB, induction among the three pancreatic cancer cell lines, except for mdm2. Further examination of other genes is necessary for a better understanding of the molecular mechanisms of enhancement of Chemosensitivity through IkB, induction. However, we have confirmed that IkB, induction leads to an increase of Chemosensitivity of pancreatic cancer. Many problems remain before clinical application of this adenoviral system will be feasible, but our results may ultimately lead to an improved therapy of pancreatic cancer. (Cancer Sci 2003; 94: 467,472) [source] | |||||||||||||||||||||||||||||||||||||