Unusual aspects of the polyamine transport system affect the design of strategies for use of polyamine analogues in chemotherapy

2007 ◽  
Vol 35 (2) ◽  
pp. 318-321 ◽  
Author(s):  
J.L.A. Mitchell ◽  
T.K. Thane ◽  
J.M. Sequeira ◽  
R. Thokala
Keyword(s):  
Regulatory Protein ◽  
Feedback System ◽  
Uptake System ◽  
Polyamine Synthesis ◽  
Polyamine Analogues ◽  
Binding Partners ◽  
Polyamine Transport ◽  
Polyamine Uptake ◽  
Tumour Cell Growth

One strategy for inhibiting tumour cell growth is the use of polyamine mimetics to depress endogenous polyamine levels and, ideally, obstruct critical polyamine-requiring reactions. Such polyamine analogues make very unusual drugs, in that extremely high intracellular concentrations are required for growth inhibition or cytotoxicity. Cells exposed to even sub-micromolar concentrations of such analogues can achieve effective intracellular levels because these compounds are incorporated by the very aggressive polyamine uptake system. Once incorporated to these levels, many of these analogues induce the synthesis of a regulatory protein, antizyme, which inhibits both polyamine synthesis and the transporter they used to enter the cell. Thus this feedback system allows steady-state maintenance of effective cellular doses of such analogues. Accordingly, effective cellular levels of polyamine analogues are generally inversely related to their capacity to induce antizyme. Antizyme activity is down-regulated by interaction with several binding partners, most notably antizyme inhibitor, and at least a few tumour tissues exhibit deficiencies in antizyme expression. Our studies explore the role of antizyme induction by several polyamine analogues in their physiological response and the possibility that cell-to-cell differences in antizyme expression may contribute to variable sensitivities to these agents.

scholarly journals Antizyme induction mediates feedback limitation of the incorporation of specific polyamine analogues in tissue culture

2004 ◽  
Vol 384 (2) ◽  
pp. 271-279 ◽  
Author(s):  
John L. A. MITCHELL ◽  
Carrie L. SIMKUS ◽  
Thynn K. THANE ◽  
Phil TOKARZ ◽  
Michelle M. BONAR ◽  
...  
Keyword(s):  
Chinese Hamster Ovary Cells ◽  
Regulatory Protein ◽  
Chinese Hamster ◽  
Feedback System ◽  
Ovary Cells ◽  
Polyamine Analogues ◽  
Mammalian Cell Growth ◽  
Steady State Levels ◽  
Uptake Activity ◽  
The Impact

Spermidine, spermine and putrescine are essential for mammalian cell growth, and there has been a pervasive effort to synthesize analogues of these polyamines that will disrupt their function and serve as tools to inhibit cell proliferation. Recently, we demonstrated that a number of such polyamine analogues are also capable of inducing the regulatory protein AZ (antizyme). In the present study the incorporation of a few sample analogues [mimics of bis(ethyl)spermine] was shown to be significantly limited by a decrease in the Vmax for the polyamine transport system in response to analogue-induced AZ. This creates an unusual circumstance in which compounds that are being designed for therapeutic use actually inhibit their own incorporation into targeted cells. To explore the impact of this feedback system, cultures of rat hepatoma HTC cells were pre-treated to exhibit either low or high polyamine uptake activity and then exposed to polyamine analogues. As predicted, regardless of initial uptake activity, all cultures eventually achieved the same steady-state levels of the cellular analogue and AZ. Importantly, analogue-induced AZ levels remained elevated with respect to controls even after the native polyamines were reduced by more than 70%. To model the insufficient AZ expression found in certain tumours, GS-CHO (GS Chinese-hamster ovary) cells were transfected to express high levels of exogenic AZI (AZ inhibitor). As anticipated, this clone incorporated significantly higher levels of the polyamine analogues examined. This study reveals a potential limitation in the use of polyamine-based compounds as therapeutics, and strategies are presented to either circumvent or exploit this elegant transport feedback system.

scholarly journals Polyamine transport is mediated by both endocytic and solute carrier transport mechanisms in the gastrointestinal tract

2010 ◽  
Vol 299 (2) ◽  
pp. G517-G522 ◽  
Author(s):  
Takeshi Uemura ◽  
David E. Stringer ◽  
Karen A. Blohm-Mangone ◽  
Eugene W. Gerner
Keyword(s):  
Carrier Transport ◽  
Cellular Functions ◽  
Synthesis Inhibitor ◽  
Polyamine Synthesis ◽  
Caveolin 1 ◽  
Polyamine Transport ◽  
Solute Carrier ◽  
Polyamine Uptake ◽  
Solute Carrier Transporter ◽  
Oxide Synthase

The polyamines spermidine and spermine, and their precursor putrescine, are required for cell growth and cellular functions. The high levels of tissue polyamines are implicated in carcinogenesis. The major sources of exogenous polyamines are diet and intestinal luminal bacteria in gastrointestinal (GI) tissues. Both endocytic and solute carrier-dependent mechanisms have been described for polyamine uptake. Knocking down of caveolin-1 protein increased polyamine uptake in colon cancer-derived HCT116 cells. Dietary supplied putrescine was accumulated in GI tissues and liver in caveolin-1 knockout mice more than wild-type mice. Knocking out of nitric oxide synthase (NOS2), which has been implicated in the release of exogenous polyamines from internalized vesicles, abolished the accumulation of dietary putrescine in GI tissues. Under conditions of reduced endogenous tissue putrescine contents, caused by treatment with the polyamine synthesis inhibitor difluoromethylornithine (DFMO), small intestinal and colonic mucosal polyamine contents increased with dietary putrescine levels, even in mice lacking NOS2. Knocking down the solute carrier transporter SLC3A2 in HCT116-derived Hkh2 cells reduced the accumulation of exogenous putrescine and total polyamine contents in DFMO treated cells, relative to non-DFMO-treated cells. These data demonstrate that exogenous putrescine is transported into GI tissues by caveolin-1- and NOS2-dependent mechanisms, but that the solute carrier transporter SLC3A2 can function bidirectionally to import putrescine under conditions of low tissue polyamines.

Role of PRAS40 in Akt and mTOR signaling in health and disease

2012 ◽  
Vol 302 (12) ◽  
pp. E1453-E1460 ◽  
Author(s):  
Claudia Wiza ◽  
Emmani B. M. Nascimento ◽  
D. Margriet Ouwens
Keyword(s):  
Mammalian Target Of Rapamycin ◽  
Mtor Signaling ◽  
Cellular Growth ◽  
S6 Kinase ◽  
Binding Partners ◽  
Health And Disease ◽  
Mtor Complex 1 ◽  
Mtor Activity

The proline-rich Akt substrate of 40 kDa (PRAS40) acts at the intersection of the Akt- and mammalian target of rapamycin (mTOR)-mediated signaling pathways. The protein kinase mTOR is the catalytic subunit of two distinct signaling complexes, mTOR complex 1 (mTORC1) and mTORC2, that link energy and nutrients to the regulation of cellular growth and energy metabolism. Activation of mTOR in response to nutrients and growth factors results in the phosphorylation of numerous substrates, including the phosphorylations of S6 kinase by mTORC1 and Akt by mTORC2. Alterations in Akt and mTOR activity have been linked to the progression of multiple diseases such as cancer and type 2 diabetes. Although PRAS40 was first reported as substrate for Akt, investigations toward mTOR-binding partners subsequently identified PRAS40 as both component and substrate of mTORC1. Phosphorylation of PRAS40 by Akt and by mTORC1 itself results in dissociation of PRAS40 from mTORC1 and may relieve an inhibitory constraint on mTORC1 activity. Adding to the complexity is that gene silencing studies indicate that PRAS40 is also necessary for the activity of the mTORC1 complex. This review summarizes the regulation and potential function(s) of PRAS40 in the complex Akt- and mTOR-signaling network in health and disease.

Role of nuclear receptors and hepatocyte-enriched transcription factors for Ntcp repression in biliary obstruction in mouse liver

2005 ◽  
Vol 289 (5) ◽  
pp. G798-G805 ◽  
Author(s):  
Gernot Zollner ◽  
Martin Wagner ◽  
Peter Fickert ◽  
Andreas Geier ◽  
Andrea Fuchsbichler ◽  
...  
Keyword(s):  
Transcription Factors ◽  
Bile Acid ◽  
Dna Binding ◽  
Nuclear Receptors ◽  
Farnesoid X Receptor ◽  
Acid Uptake ◽  
Mrna Levels ◽  
Uptake System ◽  
Duct Ligation

Expression of the main hepatic bile acid uptake system, the Na+-taurocholate cotransporter (Ntcp), is downregulated during cholestasis. Bile acid-induced, farnesoid X receptor (FXR)-mediated induction of the nuclear repressor short heterodimer partner (SHP) has been proposed as a key mechanism reducing Ntcp expression. However, the role of FXR and SHP or other nuclear receptors and hepatocyte-enriched transcription factors in mediating Ntcp repression in obstructive cholestasis is unclear. FXR knockout (FXR−/−) and wild-type (FXR+/+) mice were subjected to common bile duct ligation (CBDL). Cholic acid (CA)-fed and LPS-treated FXR−/− and FXR+/+ mice were studied for comparison. mRNA levels of Ntcp and SHP and nuclear protein levels of hepatocyte nuclear factor (HNF)-1α, HNF-3β, HNF-4α, retinoid X receptor (RXR)-α, and retinoic acid receptor (RAR)-α and their DNA binding were assessed. Hepatic cytokine mRNA levels were also measured. CBDL and CA led to Ntcp repression in FXR+/+, but not FXR−/−, mice, whereas LPS reduced Ntcp expression in both genotypes. CBDL and LPS but not CA induced cytokine expression and reduced levels of HNF-1α, HNF-3β, HNF-4α, RXRα, and RARα to similar extents in FXR+/+ and FXR−/−. DNA binding of these transactivators was unaffected by CA in FXR+/+ mice but was markedly reduced in FXR−/− mice. In conclusion, Ntcp repression by CBDL and CA is mediated by accumulating bile acids via FXR and does not depend on cytokines, whereas Ntcp repression by LPS is independent of FXR. Reduced levels of HNF-1α, RXRα, and RARα in CBDL FXR−/− mice and reduced DNA binding in CA-fed FXR−/− mice, despite unchanged Ntcp levels, indicate that these factors may have a minor role in regulation of mouse Ntcp during cholestasis.

scholarly journals Assessment of the Role of Activator Protein-1 on Transcription of the Mouse Steroidogenic Acute Regulatory Protein Gene

2004 ◽  
Vol 18 (3) ◽  
pp. 558-573 ◽  
Author(s):  
Pulak R. Manna ◽  
Darrell W. Eubank ◽  
Douglas M. Stocco
Keyword(s):  
Binding Site ◽  
Gene Transcription ◽  
Dna Sequences ◽  
Regulatory Protein ◽  
Activator Protein 1 ◽  
Transcriptional Responses ◽  
Activator Protein ◽  
Steroidogenic Acute Regulatory ◽  
Star Gene

Abstract cAMP-dependent mechanisms regulate the steroidogenic acute regulatory (StAR) protein even though its promoter lacks a consensus cAMP response-element (CRE, TGACGTCA). Transcriptional regulation of the StAR gene has been demonstrated to involve combinations of DNA sequences that provide recognition motifs for sequence-specific transcription factors. We recently identified and characterized three canonical 5′-CRE half-sites within the cAMP-responsive region (−151/−1 bp) of the mouse StAR gene. Among these CRE elements, the CRE2 half-site is analogous (TGACTGA) to an activator protein-1 (AP-1) sequence [TGA(C/G)TCA]; therefore, the role of the AP-1 transcription factor was explored in StAR gene transcription. Mutation in the AP-1 element demonstrated an approximately 50% decrease in StAR reporter activity. Using EMSA, oligonucleotide probes containing an AP-1 binding site were found to specifically bind to nuclear proteins obtained from mouse MA-10 Leydig and Y-1 adrenocortical tumor cells. The integrity of the sequence-specific AP-1 element in StAR gene transcription was assessed using the AP-1 family members, Fos (c-Fos, Fra-1, Fra-2, and Fos B) and Jun (c-Jun, Jun B, and Jun D), which demonstrated the involvement of Fos and Jun in StAR gene transcription to varying degrees. Disruption of the AP-1 binding site reversed the transcriptional responses seen with Fos and Jun. EMSA studies utilizing antibodies specific to Fos and Jun demonstrated the involvement of several AP-1 family proteins. Functional assessment of Fos and Jun was further demonstrated by transfecting antisense c-Fos, Fra-1, and dominant negative forms of Fos (A-Fos) and c-Jun (TAM-67) into MA-10 cells, which significantly (P < 0.01) repressed transcription of the StAR gene. Mutation of the AP-1 site in combination with mutations in other cis-elements resulted in a further decrease of StAR promoter activity, demonstrating a functional cooperation between these factors. Mammalian two-hybrid assays revealed high-affinity protein-protein interactions between c-Fos and c-Jun with steroidogenic factor 1, GATA-4, and CCAAT/enhancer binding protein-β. These findings demonstrate that Fos and Jun can bind to the TGACTGA element in the StAR promoter and provide novel insights into the mechanisms regulating StAR gene transcription.

scholarly journals Role of the Ndc1 interaction network in yeast nuclear pore complex assembly and maintenance

2009 ◽  
Vol 185 (3) ◽  
pp. 475-491 ◽  
Author(s):  
Evgeny Onischenko ◽  
Leslie H. Stanton ◽  
Alexis S. Madrid ◽  
Thomas Kieselbach ◽  
Karsten Weis
Keyword(s):  
Nuclear Pore Complex ◽  
Structural Integrity ◽  
Fluorescent Protein ◽  
Interaction Network ◽  
Nucleocytoplasmic Transport ◽  
Nuclear Pore ◽  
Binding Partners ◽  
Interaction Partners ◽  
Redundant Functions

The nuclear pore complex (NPC) mediates all nucleocytoplasmic transport, yet its structure and biogenesis remain poorly understood. In this study, we have functionally characterized interaction partners of the yeast transmembrane nucleoporin Ndc1. Ndc1 forms a distinct complex with the transmembrane proteins Pom152 and Pom34 and two alternative complexes with the soluble nucleoporins Nup53 and Nup59, which in turn bind to Nup170 and Nup157. The transmembrane and soluble Ndc1-binding partners have redundant functions at the NPC, and disruption of both groups of interactions causes defects in Ndc1 targeting and in NPC structure accompanied by significant pore dilation. Using photoconvertible fluorescent protein fusions, we further show that the depletion of Pom34 in cells that lack NUP53 and NUP59 blocks new NPC assembly and leads to the reversible accumulation of newly made nucleoporins in cytoplasmic foci. Therefore, Ndc1 together with its interaction partners are collectively essential for the biosynthesis and structural integrity of yeast NPCs.

scholarly journals Role of NF-κB and Myc Proteins in Apoptosis Induced by Hepatitis B Virus HBx Protein

2001 ◽  
Vol 75 (1) ◽  
pp. 215-225 ◽  
Author(s):  
Fei Su ◽  
Christian N. Theodosis ◽  
Robert J. Schneider
Keyword(s):  
Hepatitis B Virus ◽  
Hepatitis B ◽  
Apoptotic Cell ◽  
Regulatory Protein ◽  
Family Members ◽  
Necrosis Factor Alpha ◽  
Tnf Α ◽  
B Virus ◽  
High Level

ABSTRACT Chronic infection with hepatitis B virus (HBV) promotes a high level of liver disease and cancer in humans. The HBV HBx gene encodes a small regulatory protein that is essential for viral replication and is suspected to play a role in viral pathogenesis. HBx stimulates cytoplasmic signal transduction pathways, moderately stimulates a number of transcription factors, including several nuclear factors, and in certain settings sensitizes cells to apoptosis by proapoptotic stimuli, including tumor necrosis factor alpha (TNF-α) and etopocide. Paradoxically, HBx activates members of the NF-κB transcription factor family, some of which are antiapoptotic in function. HBx induces expression of Myc protein family members in certain settings, and Myc can sensitize cells to killing by TNF-α. We therefore examined the roles of NF-κB, c-Myc, and TNF-α in apoptotic killing of cells by HBx. RelA/NF-κB is shown to be induced by HBx and to suppress HBx-mediated apoptosis. HBx also induces c-Rel/NF-κB, which can promote apoptotic cell death in some contexts or block it in others. Induction of c-Rel by HBx was found to inhibit its ability to directly mediate apoptotic killing of cells. Thus, HBx induction of NF-κB family members masks its ability to directly mediate apoptosis, whereas ablation of NF-κB reveals it. Investigation of the role of Myc protein demonstrates that overexpression of Myc is essential for acute sensitization of cells to killing by HBx plus TNF-α. This study therefore defines a specific set of parameters which must be met for HBx to possibly contribute to HBV pathogenesis.

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