|ognizant Communication Corporation|
VOLUME 13, NUMBER 3
Gene Expression, Vol. 13, pp. 141-153
1052-2166/06 $90.00 + .00
Copyright © 2006 Cognizant Comm. Corp.
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Identification and Characterization of DEDD L, a Human-Specific Isoform of DEDD
Xin Huang, Minghui Zhang, Hua Tang, Chunfang Ruo, and Xuetao Cao
Institute of Immunology, Second Military Medical University, Shanghai, 200433, P.R. China
Death effector domain (DED) containing molecules are usually involved in the intracellular apoptosis cascade as executioners or regulators. One of these molecules, DEDD, was identified as a final target of the CD95 signaling pathway by which it would be transferred into the nucleolus to inhibit RNA polymerase I-dependent transcription. Here we describe a longer isoform of DEDD, DEDD L , produced by alternatively splicing, as an immune cell-specific DED-containing molecule. It is only expressed in human T lymphocytes and dendritic cells (DCs), and the mRNA expression in DCs was elevated upon inductive maturation. In cell lines MCF-7 and Jurkat, the overexpression of DEDD L could induce apoptosis more potently than that of DEDD. That DEDD L could bind FADD and cFLIP more potently than DEDD in vivo was revealed by cotransfection and immunoprecipitation. This may explain why DEDD L is a more potent apoptosis inducer, because DED-containing proteins usually induce apoptosis through DED binding. Finally, why DEDD and DEDD L are unstable in the overexpression and other studies may be explained by the finding that they are potential substrates of active caspases.
Key words: Alternative splice variant; Apoptosis; Death effector domain (DED); Dendritic cells
Address correspondence to Xin Huang, Institute of Immunology, Second Military Medical University, 800 Xiangyin Road, Shanghai 200433, P.R. China. Fax: +86-21-25070314; E-mail: firstname.lastname@example.org
Growth Arrest-Inducing Genes Are Activated in Dbl-Transformed Mouse Fibroblasts
Raffaella Melani,1 Fabio Sallustio,1 Paolo Fardin,1 Cristina Vanni,1 Marzia Ognibene,1 Catherine Ttaviano,1 Giovanni Melillo,2 Luigi Varesio,1 and Alessandra Eva1
1Laboratorio di Biologia Molecolare, Istituto G. Gaslini,
Largo G. Gaslini 5, 16147 Genova, Italy
2DTP-Tumor Hypoxia Program, National Cancer Institute, FCRF, Frederick, MD 21702, USA
The Dbl oncogene is a guanine nucleotide exchange factor for Rho GTPases and its activity has been linked to the regulation of gene transcription. Dbl oncogene expression in NIH3T3 cells leads to changes in morphological and proliferative properties of these cells, inducing a highly transformed phenotype. To gain insights into Dbl oncogene-induced transformation we compared gene expression profiles between Dbl oncogene-transformed and parental NIH3T3 cells by cDNA microarray. We found that Dbl oncogene expression is associated with gene expression modulation involving upregulation of 51 genes and downregulation of 49 genes. Five of the overexpressed genes identified are known to exert antiproliferative functions. Our observations suggest that the expression of Dbl oncogene in NIH3T3 may lead to the induction of genes associated with cell cycle arrest, possibly through the activation of stress-induced kinases.
Key words: Microarray; Dbl oncogene; Gene expression
Address correspondence to Alessandra Eva, Laboratorio di Biologia Molecolare, Istituto G. Gaslini, Largo G. Gaslini 5, 16147 Genova, Italy. Tel: +390105636633; Fax: +39010373346; E-mail: email@example.com
Posttranscriptional Control Is a Strong Factor Enabling Exclusive Expression of Surface Antigens in Paramecium tetraurelia
Martin C. Simon, Simone Marker, and Helmut J. Schmidt
Department of Biology, University of Kaiserslautern, 67663 Kaiserslautern, Germany
Variable antigens are large proteins located on the outer membrane of parasitic but also of free-living protists. Multigene families encoding surface antigens demonstrate an exclusive expression of proteins. The resulting presence of just one protein species on the cell surface is required for surface antigen function; therefore, the molecular mechanism of exclusive expression is of main interest. Regulation of gene expression and mechanisms establishing switching of antigens are hardly understood in any organism. Here we report on the reaction of Paramecium to the artificial knock down of surface antigen 51A expression by bacteria-mediated RNAi. This technique involves the feeding of dsRNA-producing bacteria. We analyzed different fragments of the target gene for dsRNA template regarding their specific knock down efficiency and found great differences. Treatment of Paramecia with RNAi against the 51A antigen demonstrated that although a massive amount of mRNA was present, the protein was not detected on the cell surface. Moreover, a minor abundance of 51D transcripts resulted in an exclusive presence of 51D proteins on the cell surface. This posttranscriptional regulation was confirmed by the transcript ratio (51A/51D) determined by real-time (RT) PCR of single cells. Because we were able to document unexclusive transcription also in wild-type cells our results indicate that this posttranscriptional regulation is a main factor of enabling exclusive gene expression. The comparison of serotype shifts, caused by efficient and inefficient knock down, indicates an involvement of full-length transcripts in regulation of gene expression. Thus, our study gives new insights into the mechanism of exclusive expression on the molecular level: (i) exclusive transcription does not occur, (ii) posttranscriptional regulation is a powerful factor enabling exclusive antigen expression, and (iii) surface antigen mRNA is shown to be involved in this mechanism in a regulating way.
Key words: Variable antigens; Exclusive expression; Posttranscriptional egulation; Silencing efficiency
Address correspondence to Martin C. Simon, Department of Biology, University of Kaiserslautern, Erwin-Schrödinger-Street, 67663 Kaiserslautern, Germany. Tel: +49-631/205-4691; Fax: +49-631/205-2496; E-mail: firstname.lastname@example.org
An Efficient Strategy to Identify Early TPA-Responsive Genes During Differentiation of HL-60 Cells
Ling-Yueh Hu,1,2 Clifford G. Tepper,3 Su-Hao Lo,4 and Wen-Chang Lin1,5
1Graduate Institute of Life Sciences, National Defense Medical
Center, Taipei, Taiwan, R.O.C.
2Institute of Biomedical Sciences, Academia Sinica, Taipei, Taiwan, R.O.C.
3Department of Biological Chemistry and Cancer Center Basic Science, University of California-Davis, Sacramento, CA 95817, USA
4Department of Orthopedic Surgery and Center for Tissue Regeneration and Repair, University of California-Davis, Sacramento, CA 95817, USA
5Institute of Bioinformatics, National Yang-Ming University, Taipei, Taiwan, R.O.C.
We have adopted a special experimental strategy to identify early responsive genes during 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced macrophage-like differentiation of human myeloid leukemia cells (HL-60). This was performed in cells that were synchronized by nocodazole and treated with TPA in the presence of a protein synthesis inhibitor, cycloheximide, to prevent activation of secondary targets and therefore increase the probability of early transcripts in total RNA pool. The expression alteration was analyzed by microarray and the selection criteria of candidate genes were adjusted by real-time PCR validation to increase its reliability. Finally, 56 genes were identified as early TPA-responsive genes in this multiscreening step approach. Furthermore, upregulation of three candidate genes (NFIL3, SKIL, and JMJD3) was shown to be dosage and time dependent with TPA treatment and was found to be directly regulated by TPA through PKC-dependent signaling. These results revealed that our screenings provide a useful and efficient approach to identify early TPA-responsive genes and these genes might involve the regulation of TPA-induced differentiation program of HL-60 cells as primary targets.
Key words: Early responsive gene; TPA; Cycloheximide; Differentiation; Microarray
Address correspondence to Wen-chang Lin, Institute of Biomedical Sciences, Academia Sinica, Taipei 115, Taiwan, R.O.C. Tel: +(886) 2-26523967; Fax: +(886) 2-27827654; E-mail: email@example.com
Gene Expression Profile of B16(F10) Murine Melanoma Cells Exposed to Hypoxic Conditions In Vitro
Magdalena Olbryt,1 Michal Jarzab,1 Joanna Jazowiecka-Rakus,2 Krzysztof Simek,3 Stanislaw Szala,2 and Aleksander Sochanik2
1Department of Tumor Biology, Maria Sklodowska-Curie Memorial
Cancer Center and Institute of Oncology, Gliwice Branch, Poland
2Department of Molecular Biology, Maria Sklodowska-Curie Memorial Cancer Center and Institute of Oncology, Gliwice Branch, Poland
3Faculty of Automatic Control, Electronics and Computer Science, Silesian University of Technology, Gliwice, Poland
Hypoxia is an important feature of tumor microenvironment, exerting far-reaching effects on cells and contributing to cancer progression. Previous studies have established substantial differences in hypoxia response between various cell lines. Investigating this phenomenon in melanoma cells contributes to a better understanding of cell lineage-specific hypoxia response and could point out novel hypoxia-regulated genes. We investigated transcriptional activity of B16(F10) murine melanoma cells cultured for 24 h under hypoxic (nominal 1% O2, 15 samples including controls) and hypoxia-mimicking conditions (cobalt chloride, 100 or 200 mM, 6 samples including controls). Gene expression profiles were analyzed using MG-U74Av2 oligonucleotide microarrays. Data analysis revealed 2541 probesets (FDR <5%) for 1% oxygen experiment and 364 probesets (FDR <5%) for cobalt chloride, which showed differences in expression levels. Analysis of hypoxia-regulated genes (true hypoxia, 1% O2) by stringent Family-Wise Error Rate estimation indicated 454 significantly changed transcripts (p < 0.05). The most upregulated genes were Lgals3, Selenbp1, Nppb (more than ten-fold increase). We observed significant differences in expression levels of genes regulating glycolysis (Pfkp, Hk2, Aldo3, Eno2), apoptosis (Bnip3, Bnip3l, Cdkn1a), transcription (Bhlhb2, Sap30, Atf3, Mxi1), angiogenesis (Vegfa, Adm, Anxa2, Ctgf), adhesion (Pkp2, Itga4, Mcam), migration (Cnn2, Tmsb4x), and other processes. Both true hypoxia and hypoxia mimicry induced HIF-1-regulated genes. However, unsupervised analysis (Singular Value Decomposition) revealed distinct differences in gene expression between these two experimental conditions. Contrary to hypoxia, cobalt chloride caused suppression of gene expression rather than stimulation, especially concerning transcripts related to proliferation, immune response, DNA repair, and melanin biosynthesis.
Key words: Hypoxia; Cobalt chloride; Melanoma; Oligonucleotide microarrays; Singular Value Decomposition
Address correspondence to Magdalena Olbryt, Department of Tumor Biology, Maria Sklodowska-Curie Memorial Cancer Center and Institute of Oncology, Gliwice Branch, Wybrzeze Armii Krajowej 15, 44-101 Gliwice, Poland. Tel: 48-32-278 9888; Fax: 48-32-231 3512; E-mail: firstname.lastname@example.org
Multiregional Gene Expression Profiling Identifies MRPS6 as a Possible Candidate Gene for Parkinson's Disease
Spiridon Papapetropoulos,1 Jarlath Ffrench-Mullen,2 Donald M C Corquodale,1 Yujing Qin,1 John Pablo,1 and Deborah C. Mash1
1Department of Neurology, University of Miami, Miller School
of Medicine, Miami, FL, USA
2Gene Logic Inc., Gaithersburg, MD, USA
Combining large-scale gene expression approaches and bioinformatics may provide insights into the molecular variability of biological processes underlying neurodegeneration. To identify novel candidate genes and mechanisms, we conducted a multiregional gene expression analysis in postmortem brain. Gene arrays were performed utilizing Affymetrix HG U133 Plus 2.0 gene chips. Brain specimens from 21 different brain regions were taken from Parkinson's disease (PD) (n = 22) and normal aged (n = 23) brain donors. The rationale for conducting a multiregional survey of gene expression changes was based on the assumption that if a gene is changed in more than one brain region, it may be a higher probability candidate gene compared to genes that are changed in a single region. Although no gene was significantly changed in all of the 21 brain regions surveyed, we identified 11 candidate genes whose pattern of expression was regulated in at least 18 out of 21 regions. The expression of a gene encoding the mitochondria ribosomal protein S6 (MRPS6) had the highest combined mean fold change and topped the list of regulated genes. The analysis revealed other genes related to apoptosis, cell signaling, and cell cycle that may be of importance to disease pathophysiology. High throughput gene expression is an emerging technology for molecular target discovery in neurological and psychiatric disorders. The top gene reported here is the nuclear encoded MRPS6, a building block of the human mitoribosome of the oxidative phosphorylation system (OXPHOS). Impairments in mitochondrial OXPHOS have been linked to the pathogenesis of PD.
Key words: Parkinson's disease; Mitoribosome; MRPS6; Microarray; Postmortem
Address correspondence to Deborah C. Mash, Ph.D., Departments of Neurology and Molecular and Cellular Pharmacology, University of Miami, Miller School of Medicine, 1501 NW 9th Avenue (NPF), Miami, FL 33136, USA. Tel: (+1) (305) 243-5888; Fax: (+1)(305) 243-3649; E-mail: email@example.com