Functional Myc-Max heterodimer is required for activation-induced apoptosis in T cell hybridomas.
01 Dec 1994-Journal of Experimental Medicine (Rockefeller University Press)-Vol. 180, Iss: 6, pp 2413-2418
TL;DR: It is shown that coexpression of a reciprocally mutant Myc protein capable of forming functional heterodimers with the mutant Max can compensate for the dominant negative activity and restore activation-induced apoptosis.
Abstract: T cell hybridomas respond to activation signals by undergoing apoptotic cell death, and this is likely to represent comparable events related to tolerance induction in immature and mature T cells in vivo. Previous studies using antisense oligonucleotides implicated the c-Myc protein in the phenomenon of activation-induced apoptosis. This role for c-Myc in apoptosis is now confirmed in studies using a dominant negative form of its heterodimeric binding partner, Max, which we show here inhibits activation-induced apoptosis. Further, coexpression of a reciprocally mutant Myc protein capable of forming functional heterodimers with the mutant Max can compensate for the dominant negative activity and restore activation-induced apoptosis. These results imply that Myc promotes activation-induced apoptosis by obligatory heterodimerization with Max, and therefore, by regulating gene transcription.
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01 Jan 2003
TL;DR: The established activation of the survivin promoter provides a molecular explanation for its oncogenic involvement, and the differential activities toward DEC2 and survivin promoters establish that DEC I can act as a repressor or an activator depending on the genomic context of a target gene.
Abstract: The basic helix-loop-helix (bHLH) proteins are int imately associated with developmental events such as cell differentiation and lineage commitment. The HLH domain in the bHLH motif is responsible for dimerization whereas the basic region mediates sequence specific DNA binding. Human DEC, mouse STRA and rat SHARP proteins represent a new c lass of bHLH proteins. In each species, two members are identified wi th a sequence identi ty of>90% in the bHLH region and 40% in the total protein, respectively. Based on sequence alignment and domain analysis, DEC/STRA/SHARP proteins show high s imilarities (40%) to Drosoph ila Hairy and E(spl) as well as the mammalian homologues (e.g., HES) in the bHLH domain. However, they lack the C-terminal WRPW motif which is present at all other Ha iry/E(spl)/Hes proteins and mediates transcription repression. These structural d ifferences distinguish DEC/STRA/SHARPs from other bHLH proteins and indicate that they have dist inct biological functions. The purpose of this dissertation is to elucidate the oncogenic roles of DEC I and determine the molecular actions of DEC I on transcription regulation. Expression measurments demonstrate that DEC I is abundantly expressed in cancer tissues but not in adjacent nonmal tissues. In stable transfectants, DEC I inhibits ce ll proliferation, antagonizes serum deprivation-induced apoptosis, and selectively decreases the activities of several major caspases. Western blott ing analyses identify that antiapoptot ic protein survivin is a fu nctional mediator responsible for DEC I-directed antiapoptotic activity. DEC I and survivin exhibit a paralleled expression pattern in paired tumor-normal tissues. In co-transfection experiments, DEC I stimulates the survivin promoter, and this mechanism relies on the physical interactions with Sp I sites in the proximal promoter. In contrast, DEC I and its structurally related protein DEC2 show an inverted expression pattern in paired tumor-normal tissues. Forced expression of DEC I causes proportional decreases in the expression of DEC2 in stable transfectants. Co-transfection with DEC I represses the activity of a DEC2 promoter reporter by as much as 90%. The DEC I mediated transcription repression is achieved by direct binding to the E-box element in the proximal promoter of DEC2. The established activation of the survivin promoter provides a molecular explanation for its oncogenic involvement, and the differential activities toward DEC2 and survivin promoters establish that DEC I can act as a repressor or an activator depending on the genomic context of a target gene. ACKNOWLEDGEMENT This dissertation is a culmination of several years of work, during which time, I have greatly benefited from interactions wi th a great many friends, teachers, and family members who have taught me, encouraged me and supported me. I would first like to express my s incere gratitude to my mentor Dr. Bingfang Yan for his leadership, discipline and knowledge that guided me throughout this endeavor. I consider mysel f extremely fortunate to have worked with him. I would also like to express my sincere appreciation to the members of my dissertation commettee, Ors Nasser H. Zawia, Gongqin Sun, Kayvous Parang, and Fatem Akahlaghi, for their time, their consideration and their valuable suggestions. I would also thank all of the past the present members of Dr. Yan 's research group for their friendship, camaraderie and readiness to help. Special thanks to Dr. He Zhang for his friendship and help, to Dr. Mingxing Xie for conducting the western blots described in manuscript Ill , to Dr. Xiulong Song for cloning the mutant reporters described in manuscript IJI and to Dr. Ryaz Mohammad fo r his help with the gel shift assay. Finally, I wou ld like to thank my wife Ying Wang. She supported me throughout my whole study period. I would also like to thank to my parents and my parents-in-law, for their love, support and encouragement. TABLE OF CONTENTS ABSTRACT ....... ... .. . .. . . . . .... . ....... . ..... . . ..... ii ACKNOWLEDGEMENT. . . .iv TABLE OF CONTENTS . ......... ......... . . ....... v LIST OF TABLES ... LIST OF FIGURES . . ..•..•.... . .. ..
2 citations
01 Jan 1997
TL;DR: The aim of this work is to determine the signaling pathways in response to chemotherapeutic agents with the goal of developing more effective drugs and new combinations of existing drugs.
Abstract: Many diseases including cancer arise from alterations in signaling pathways. The cellular machinery controlling whether a cell is quiescent, proliferates, differentiates, or dies is no longer “correctly” regulated in transformed cells.1 However, some cancer cells retain the ability to undergo differentiation and cell death (primarily those cells of the hematopoietic and immune systems). The signaling mechanisms by which current anticancer agents induce differentiation and cell death are not completely understood. However, because of their central role in controlling cell growth and differentiation these signaling pathways are attractive targets for the improvement of therapy regimens and the development of new drugs.2,3 It is our aim to determine the signaling pathways in response to chemotherapeutic agents with the goal of developing more effective drugs and new combinations of existing drugs.
1 citations
Dissertation•
01 Jan 1997
1 citations
Patent•
14 Mar 2014TL;DR: In this article, T cells expressing artificial cell death polypeptides that cause death of a cell, e.g., T lymphocytes, were used to treat diseases such as cancer.
Abstract: Provided herein are cells, e.g., T cells expressing artificial cell death polypeptides that cause death of a cell, e.g., cells (e.g., T lymphocytes) expressing the cell death polypeptide, when the cell death polypeptide is multimerized or dimerized. Also provided herein is use of such cells, e.g., T lymphocytes, to treat diseases such as cancer.
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Journal Article•
TL;DR: This highly reproducible, quantitative assay for T cell growth factor (TCGF), based upon the tritiated-thymidine incorporation of continuous murine tumor-specific cytotoxic T cell lines (CTLL), has revealed that T lymphocytes are required for its production.
Abstract: Several soluble factors have recently been associated with the proliferation and differentiation of thymus-derived lymphocytes. One of these factors present in medium conditioned by T cell mitogen-stimulated lymphocytes has the ability to promote the long-term culture of normal and antigen-specific cytotoxic T cells. We report a method to test for this proliferative stimulus in the form of a sensitive microassay based upon the tritiated thymidine incorporation of continuous murine tumorspecific cytotoxic T cell lines (CTLL). The microassay requires microliter quantities of sample fluid and is amenable to quantitative analysis. This highly reproducible, quantitative assay for T cell growth factor (TCGF) has allowed investigation as to the kinetics of TCGF generation and has revealed that T lymphocytes are required for its production. Further investigation has supported the notion that this nonspecies-specific factor is actively removed from tissue culture medium by the proliferation of either T cell mitogen-activated lymphocytes or CTLL.
3,106 citations
TL;DR: It is demonstrated that deregulated c-myc expression induces apoptosis in cells growth arrested by a variety of means and at various points in the cell cycle.
3,047 citations
TL;DR: The present review describes several methods to characterize and differentiate between two different mechanisms of cell death, apoptosis and necrosis, applied to studies of apoptosis triggered in the human leukemic HL-60 cell line by DNA topoisomerase I or II inhibitors, and in rat thymocytes by either topoisomersase inhibitors or prednisolone.
Abstract: The present review describes several methods to characterize and differentiate between two different mechanisms of cell death, apoptosis and necrosis. Most of these methods were applied to studies of apoptosis triggered in the human leukemic HL-60 cell line by DNA topoisomerase I or II inhibitors, and in rat thymocytes by either topoisomerase inhibitors or prednisolone. In most cases, apoptosis was selective to cells in a particular phase of the cell cycle: only S-phase HL-60 cells and G0 thymocytes were mainly affected. Necrosis was induced by excessively high concentrations of these drugs. The following cell features were found useful to characterize the mode of cell death: a) Activation of an endonuclease in apoptocic cells resulted in extraction of the low molecular weight DNA following cell permeabilization, which, in turn, led to their decreased stainability with DNA-specific fluorochromes. Measurements of DNA content made it possible to identify apoptotic cells and to recognize the cell cycle phase specificity of the apoptotic process. b) Plasma membrane integrity, which is lost in necrotic but not apoptotic cells, was probed by the exclusion of propidium iodide (PI). The combination of PI followed by Hoechst 33342 proved to be an excellent probe to distinguish live, necrotic, early- and late-apoptotic cells. c) Mitochondrial transmembrane potential, assayed by retention of rhodamine 123 was preserved in apoptotic but not necrotic cells. d) The ATP-dependent lysosomal proton pump, tested by the supravital uptake of acridine orange (AO) was also preserved in apoptotic but not necrotic cells. e) Bivariate analysis of cells stained for DNA and protein revealed markedly diminished protein content in apoptotic cells, most likely due to activation of endogenous proteases. Necrotic cells, having leaky membranes, had minimal protein content. f) Staining of RNA allowed for the discrimination of G0 from G1 cells and thus made it possible to reveal that apoptosis was selective to G0 thymocytes. g) The decrease in forward light scatter, paralleled either by no change (HL-60 cells) or an increase (thymocytes) of right angle scatter, were early changes during apoptosis. h) The sensitivity of DNA in situ to denaturation, was increased in apoptotic and necrotic cells. This feature, probed by staining with AO at low pH, provided a sensitive and early assay to discriminate between live, apoptotic and necrotic cells, and to evaluate the cell cycle phase specificity of these processes. i) The in situ nick translation assay employing labeled triphosphonucleotides can be used to reveal DNA strand breaks, to detect the very early stages of apoptosis.(ABSTRACT TRUNCATED AT 400 WORDS)
1,953 citations
TL;DR: In this paper, the Max polypeptide when associated with the Myc or Mad polyPEptide is capable of binding to nucleotide sequences containing CACGTG.
Abstract: Nucleic acid molecules capable of hybridizing under stringent conditions to the nucleotide sequence residing between positions 1 and 453 of the max cDNAs shown in Figure 2, or to the nucleotide sequence reisiding between positions 148 and 810 of the mad cDNAs shown in Figure 14. The Max polypeptide when associated with the Myc or Mad polypeptide is capable of binding to nucleotide sequences containing CACGTG.
1,602 citations
TL;DR: Results identify T3-epsilon as a cell surface protein involved in the transduction of activation signals and can both activate and inhibit T-cell function.
Abstract: A monoclonal antibody (145-2C11) specific for the murine T3 complex was derived by immunizing Armenian hamsters with a murine cytolytic T-cell clone. The antibody is specific for a 25-kDa protein component (T3-epsilon) of the antigen-specific T-cell receptor. It reacts with all mature T cells and can both activate and inhibit T-cell function. These results identify T3-epsilon as a cell surface protein involved in the transduction of activation signals.
1,509 citations