|ognizant Communication Corporation|
VOLUME 8, NUMBER 5-6
Gene Expression, Vol. 8, pp. 263-272, 1999
1052-2166/99 $20.00 + .00
Copyright © 1999 Cognizant Comm. Corp.
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Kwan Y. Thai and James D. Forney
Department of Biochemistry, 1153 Biochemistry Building, Purdue University, West Lafayette, IN 47907
Variable surface antigens are commonly found on free-living and parasitic protozoa, yet the regulation of antigen expression and switching is not fully understood in any system. A cell line of Paramecium tetraurelia stock 51 can express at least 11 different antigens yet only one type is found on the surface at any one time. Previous studies have shown that mutually exclusive expression of Paramecium surface antigens can be overcome if two antigen genes contain the same 5' coding region. In this article we utilize a gene chimera containing portions of A51 and B51 to analyze the effect of a frameshift mutation on transcription and steady-state mRNA levels. We show that a frameshift mutation near the 3' end prevents expression of the protein on the cell surface and reduces the rate of transcription of the corresponding gene. The difference in transcription is not the result of differences in plasmid copy number. We propose that expression of the antigen on the cell surface is part of a self-regulatory pathway that influences transcription of the corresponding gene. A model incorporating the previous and current data is presented.
Key Words: Transcription; Variable surface antigen; Paramecium; GPI-anchored protein
Address correspondence to James Forney, 1153 Biochemistry Building, West Lafayette, IN 47907-1153. Tel: (765) 494-1632; Fax: (765) 494-7897; E-mail: firstname.lastname@example.org
Mohan B. Kumar1 and Gary H. Perdew1,2
1Graduate Program in Biochemistry and Molecular Biology, and 2Center for Molecular Toxicology and the Department of Veterinary Science, The Pennsylvania State University, University Park, PA 16802
The aryl hydrocarbon receptor (AhR), a soluble cytosolic protein, mediates many of the toxic effects of TCDD and related chemicals. The toxic effects are largely cell, tissue, and promoter context dependent. Although many details of the overall dioxin signal transduction have been elucidated, the transcriptional regulation of dioxin-induced genes like cyp1A1 is not yet completely understood. Previously, we have shown that the co-regulator RIP140 is a potential AhR coactivator. In this report, the role of coactivator, SRC-1, in AhR-mediated transcriptional regulation was examined. SRC-1 increased AhR-mediated, TCDD-dependent reporter gene activity threefold in Hepa-1 and COS-1 cells. In in vitro interaction assays, SRC-1 was found to interact with AhR but not with ARNT. SRC-1 interacted weakly with AhR in the absence of TCDD and the addition of ligand further increased SRC-1 binding to AhR. Deletional mapping studies of the AhR revealed that SRC-1 binds to the AhR transactivation domain. Finer mapping of the SRC-1-interacting subdomains in the AhR transactivation domain suggested that the Q-rich subdomain was necessary and sufficient for interaction, similar to that seen with RIP140. Using GFP-tagged constructs, SRC-1 was shown to interact with AhR in cells. Unlike RIP140, LXXLL motifs in SRC-1 were necessary for interaction with AhR in vitro and for coactivation in Hepa-1 cells. The recruitment of certain coactivators by a variety of receptors suggests possible common coactivator pools and competition among receptors for limiting coactivators. Examination of the role of SRC-1 in AhR/ARNT transactivation in ARNT-deficient mutant Hepa-1 c4 cells demonstrates that the AhR transactivation domain is sufficient for enhanced coactivation mediated by SRC-1 in the presence of a transactivation domain deleted ARNT protein.
Key Words: Ary hydrocarbon receptor (AhR); Dioxin; Coactivator; SRC-1
Address correspondence to Gary H. Perdew, Department of Veterinary Science, 115 Henning Building, University Park, PA 16802. Tel: (814) 865-0400. (814) 863-6140; E-mail: email@example.com
Y. Higashimoto,1 N. Keicho,2 W. M. Elliott,1 J. C. Hogg,1 and S. Hayashi1
1University of British Columbia Pulmonary Research Laboratory,
Vancouver, BC V6Z 1Y6 Canada
2Department of Respiratory Medicine, University of Tokyo, 7-3-1 Hongo Bunkyo-ku, Tokyo, 113-8655 Japan
In previous studies we demonstrated that the E1A DNA and proteins of group C adenovirus are present in excess in the lungs of patients with chronic obstructive pulmonary disease (COPD). Because adenovirus E1A gene products are known to regulate the expression of many genes by interacting with cellular transcription factors, we postulated that E1A enhances the production of inflammatory mediators and exacerbates the inflammatory process in smokers' lungs. We reported that LPS-induced ICAM-1 expression in A549 cells is upregulated by E1A. In the current study we investigated whether this regulation is mediated through the ICAM-1 promoter. A549 cells and primary human bronchial epithelial (HBE) cells were transiently cotransfected with a plasmid containing the ICAM-1 enhancer-promoter linked to the chloramphenicol acetyltransferase (CAT) reporter gene (pBS-CAT-P) and either a plasmid carrying the adenovirus 5 E1A gene (pE1Aneo) or a control plasmid (pneo). To compare the effect of transient versus stable E1A expression on the activity of this promoter, we also transiently transfected stable E1A-expressing A549 cells with pBS-CAT-P. Transient cotransfection of pE1Aneo and pBS-CAT-P had no effect on basal ICAM-1 promoter activity in A549 or HBE cells. After stimulation of A549 cells with TNF-a, IFN-g, or LPS, promoter activity was increased by two- to threefold in the presence of adenovirus E1A. In HBE cells, on the other hand, E1A repressed the ICAM-1 promoter after stimulation with IFN-g and LPS with little change after TNF-a stimulation. In stable E1A transfectants, ICAM-1 promoter activity was 2 to 2.5 times higher than in control transfectants with or without stimulation with TNF-a or LPS. These findings suggest that E1A can modulate the activity of the ICAM-1 promoter in lung epithelial cells and this modulation is different in cells of alveolar origin compared to bronchial epithelial cells.
Key Words: Adenovirus E1A; Lung epithelial cells; ICAM-1 promoter; Inflammatory stimuli
Address correspondence to Shizu Hayashi, U.B.C. Pulmonary Research Laboratory, St. Paul's Hospital, 1081 Burrard Street, Vancouver, BC Canada. Tel: (604) 806-8346; Fax: (604) 806-8351; E-mail: firstname.lastname@example.org
Joé Attal, Marie-Claire Theron, Claudine Puissant, and Louis Marie Houdebine
Unité de Différenciation Cellulaire, Institut National de la Recherche Agronomique, 78352 Jouy en Josas, Cedex France
Specific structures found in the mRNA of picornavirus are known to allow a cap-independent translation. These structures, named internal ribosome entry sites (IRES), are also able to favor translation of the second cistron in bicistronic mRNAs. Their mechanism of action is not well understood. In the present study, two IRESs have been used: the IRES from poliovirus and a newly discovered IRES (SUR) composed of the 5' P untranslated sequence from SV40 early genes, the R structure, and a small part of the U5 region from the human leukemia virus-1 (HTLV-1). The bicistronic constructs containing the firefly luciferase gene as the first cistron and the chloramphenicol acetyltransferase (CAT) as the second cistron were driven by the Rous sarcoma virus (RSV) promoter and contained the early gene SV40 terminator. All the resulting plasmids were tested by transfection in HeLa and CHO cells. In the bicistronic mRNAs without IRES, the expression of the CAT gene was dependent on the distance between the two cistrons. The maximum efficiency in the expression of the second cistron was obtained when the intercalating RNA was composed of 30 to 90 nucleotides. This expression was deeply reduced when the intercalating fragment contained 8 or 300 nucleotides and was undetectable with 500 nucleotides. Unexpectedly, the luciferase mRNA was almost not expressed when the intercalating RNA was of 8 or 30 nucleotides. Expression of the luciferase gene occurred when the intercistronic RNA fragment was of 80 nucleotides and it became lower at 300 and 500 nucleotides. The same observations were done when the poliovirus or the SUR IRESs were added after the intercistronic spacers. However, expression of the CAT gene was amplified by both IRESs. When the CAT cistron preceded by the poliovirus or SUR IRES was introduced within luciferase cistron, 316 nucleotides before its termination codon, the IRESs were able to initiate translation of the following CAT gene irrespectively of the mRNA luciferase reading frame. Moreover, with all these constructs the highest expression level of the CAT cistron did not exceed 10% of that obtained with the same vector carrying only the CAT cistron. To identify a possible relation between the IRESs and the cap site, the CAT cistron preceded or not with an IRES was introduced 210 nucleotides downstream of the AUG codon of the luciferase gene (i.e., 258 nucleotides from the cap site) and 100 nucleotides after an added UAG termination codon. Expression of the CAT gene was not modified by the addition of the poliovirus IRES but it was strongly stimulated by the SUR IRES (the level of expression corresponded to 65% of that obtained with the same vector carrying only the CAT cistron). These results suggest that there is a cooperation between the cap and the SUR IRES and not the poliovirus IRES to stimulate translation. These data indicate that IRESs must be introduced in precise position to allow an efficient expression of the second cistron in bicistronic mRNAs.
Key Words: Internal ribosome entry site (IRES); Translation; Bicistronic mRNA
Address correspondence to Louis Marie Houdebine, Unité de Différenciation Cellulaire, Institut National de la Recherche Agronomique, 78352 Jouy en Josas, Cedex France. Tel: 33 1 34 65 25 40; Fax: 33 1 34 65 22 41; E-mail: email@example.com
Amanda Capes-Davis, Scott D. Andrew, Valentine J. Hyland, Stephen Twigg, Diana L. Learoyd, Trisha Dwight, Debbie J. Marsh, and Bruce G. Robinson
Kolling Institute of Medical Research and Department of Endocrinology, Royal North Shore Hospital, University of Sydney, Sydney, N.S.W., Australia
The RET proto-oncogene encodes a receptor tyrosine kinase activated by the binding of factors from the glial cell line-derived neurotrophic factor (GDNF) family to receptor-a components such as GDNF family receptor a-1 (GFRa-1). Mutations within the sequence of the RET proto-oncogene are associated with multiple endocrine neoplasia type 2 (MEN 2), an inherited tumor syndrome characterized by the development of medullary thyroid carcinoma (MTC) and other neuroendocrine tumors. Despite Northern analysis showing that RET is expressed in the majority of MTCs, the factors regulating this expression are poorly understood. To address this issue we examined RET expression in response to glucocorticoids in the TT cell line, derived from a metastatic MTC. The synthetic glucocorticoid dexamethasone was found to reduce RET expression at both mRNA and protein levels. This effect was dose responsive and maximal at 24 h. The reduction in RET mRNA was shown to be specific to glucocorticoids and was also seen in a primary MTC culture. Nuclear run-on studies revealed the reduction in steady-state RNA to be due to a decrease in RET mRNA transcription and the effect was shown to be independent of new protein synthesis or RNA stability. Dexamethasone was also found to exert an inhibitory effect upon cell growth, suggesting a potential use for glucocorticoids in the treatment of medullary carcinoma and MEN 2.
Key Words: RET proto-oncogene; Multiple endocrine neoplasia type 2; Gene expression; Alternative splicing; Dexamethasone
Address correspondence to Professor B. G. Robinson, Kolling Institute of Medical Research, Royal North Shore Hospital, St Leonards, 2065 N.S.W., Australia. Tel: +61 2 9926 7267; Fax: +61 2 9926 8523; E-mail: firstname.lastname@example.org
Xiao Li Sun,1,4 Dawei Li,2 Jin Fang,1,3 Bruce Casto,3 Inge Noyes,4 and George E. Milo1,4
1Department of Medical Biochemistry and Comprehensive Cancer
Center, Comprehensive Cancer Center, 410 W. 12th Avenue, The Ohio State
University College of Medicine and Public Health, Columbus, OH
2Harvard University, College of Medicine, Boston, MA
3Division of Environmental Health Sciences, School of Public Health, The Ohio State University, Columbus, OH 43210
4Center for Molecular and Environmental Health, The Ohio State University, Columbus, OH
A linear model for human cell metastasis has been developed in vitro from chemically transformed normal human cells. The chemically transformed cells are nontumorigenic in nude mice, but can be converted to a tumorigenic phenotype by transfection with a nondirectional cDNA library or antisense cDNA to the ML-1 gene. The primary transfected cell line (TR1T) forms localized, progressively growing tumors in nude mice that do not invade into the surrounding tissue. This tumorigenic TR1T cell line could be advanced into a metastatic stage following an additional transfection (TR2M cell line) with the cDNA expression library or antisense cDNA to the ML-1 gene. Metastatic cells, selected from tumors that were attached to internal organs, exhibited an increase in invasiveness as measured in vitro using an invasion chamber. The metastatic cells also exhibited an increased expression of matrix metalloproteinase-1 (MMP-1), although MMP-1 was not part of the cDNA that was transfected into either the TR1T cells or the doubly transfected metastatic TR2M cells. These data suggest that the increase in MMP-1 expression was a secondary downstream event responding to an upstream genetic change that initiated the conversion of cells from a tumorigenic to a metastatic stage. In summary, human cell lines representing premalignant, malignant, and metastatic phenotypes have been established in culture that can be used to identify gene changes that occur as normal human cells progress to a metastatic stage during tumor development. One gene, ML-1, that is found in the expression library appears to be involved in malignant progression, because ML-1 antisense cDNA will convert chemically transformed cells to both tumorigenic and metastatic stages, and cells from both local and metastatic tumors have a reduced or complete loss of expression of the ML-1 gene.
Key Words: Metastasis; cDNA transfection; Matrix metalloproteinase
Address correspondence to Xiao Li Sun, Ph.D., M.D., Comprehensive Cancer Center, The Ohio State University, 410 W. 12th Avenue, Room 402, Columbus, OH 43210. Tel: (614) 292-3406; Fax: (614) 292-1400; E-mail: email@example.com
Ryuichi Nakashima,1,2 Huijuan Song,1 Takayuki Enomoto,2 Yuji Murata,2 Michael R. McClaid,1 Bruce C. Casto,1 and Christopher M. Weghorst1
1Division of Environmental Health Sciences, School of Public
Health, College of Medicine and Public Health and the Comprehensive Cancer
Center, The Ohio State University, Columbus, OH 43210-1240
2Department of Obstetrics and Gynecology, Osaka University Faculty of Medicine, Osaka, Japan 565-0871
Cellular responses to the transforming growth factor b (TGFb) ligand, including inhibition of cell proliferation, are mediated by a heteromeric receptor complex composed of TGFb types I and II receptors (TbR-I and TbR-II). Loss of responsiveness to TGFb, attributed to inactivation of the TbR complex, has been implicated in the development of tumors in a number of human epithelial and lymphoid tissues. To gain a better understanding of TGFb signal transduction pathways in endometrial carcinogenesis, we have investigated the role of the TbR complex by evaluating the TbR-I and TbR-II genes for mutations throughout the entire coding region in human sporadic endometrial tumors. Using reverse transcription-PCR, "Cold" single-strand conformation polymorphism analysis, and direct DNA sequencing, it was found that 1 of 39 (2.6%) and 7 of 42 samples (17%) contained code-altering changes in the kinase domain of TbR-I and TbR-II, respectively. In TbR-I, a 3-bp deletion was found resulting in replacement of Arg and Glu at codon 237 and 238 by Lys. With TbR-II, mutations were found in the kinase, the extracellular, and the C-terminal domains. No frameshift mutations were detected; however, a silent population polymorphism (AAC-->AAT at codon 389) in TbR-II was found in 19 of 42 (44%) tumor samples. These results suggest that alteration in TbR-II, but not TbR-I, has an important role in the development of endometrial carcinoma.
Key Words: TGFb receptors; Endometrial carcinoma; Mutational analysis; "Cold" SSCP
Address correspondence to Christopher M. Weghorst, Ph.D., 1148 James
CHRI, The Ohio State University, Division of Environmental Health Sciences,
College of Medicine and Public Health, 300 W. 10th Ave., Columbus, OH 43210-1240.
Tel: (614) 293-3713; Fax: (614) 293-3333; E-mail: weghorst.2(@osu.edu