[Show abstract][Hide abstract] ABSTRACT: Interactions with cofactors regulate transcriptional activity and also help HOX proteins to achieve the specificity required for transcriptional regulation of target genes. In this study, we describe a novel protein/protein interaction of HOXB7 with poly (ADP-ribose) polymerase-1 (PARP-1) that involves the homeodomain of HOXB7 and the first zinc finger domain of PARP-1. Upon binding to PARP-1, HOXB7 undergoes poly(ADP-ribosyl)altion resulting in a reduction of its transcriptional activity. Since aspartic acid and glutamic acid residues are acceptors of the ADP ribose moiety transferred by PARP-1, deletion of the evolutionarily conserved C-terminal Glu-rich tail of HOXB7 dramatically attenuates ADP-ribosylation of HOXB7 by PARP-1. Further, a mutant of HOXB7 without the Glu-rich tail loses the ability to be negatively regulated by PARP-1 and becomes transcriptionally more active in luciferase reporter assays. Since the homeodomain is highly conserved among HOX proteins, five other HOX proteins were tested. All six showed interaction with, and were poly(ADP-ribosyl)ated by PARP-1. However, among them, this modification altered the DNA binding activity of only HOXA7 and HOXB7. In summary, this study identifies a new interacting partner of HOX proteins. More importantly, this study reveals a novel mechanism whereby polyADP-ribosylation regulates transcriptional activities of HOX proteins such as HOXB7 and HOXA7.
PLoS ONE 07/2012; 7(7):e40644. DOI:10.1371/journal.pone.0040644 · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The packaging of DNA into nucleosomes imposes obstacles on gene transcription, and histone-modifying and nucleosome-remodeling complexes work in concert to alleviate these obstacles so as to facilitate transcription. Emerging evidence shows that chromatin-associated poly(ADP-ribose) polymerase 1 (PARP-1) and its enzymatic activity facilitate inflammatory gene transcription and modulate the inflammatory response in animal models. However, the molecular mechanisms by which PARP-1 enzymatic activity facilitates transcription are not well understood. Here we show that through an intracellular signaling pathway, lipopolysaccharide (LPS) stimulation induces PARP-1 enzymatic activity and the ADP-ribosylation of histones at transcriptionally active and accessible chromatin regions in macrophages. In vitro DNase I footprinting and restriction endonuclease accessibility assays reveal that histone ADP-ribosylation directly destabilizes histone-DNA interactions in the nucleosome and increases the site accessibility of the nucleosomal DNA to nucleases. Consistent with this, LPS stimulation-induced ADP-ribosylation at the nucleosome-occupied promoters of il-1β, mip-2, and csf2 facilitates NF-κB recruitment and the transcription of these genes in macrophages. Therefore, our data suggest that PARP-1 enzymatic activity facilitates gene transcription through increasing promoter accessibility by histone ADP-ribosylation.
[Show abstract][Hide abstract] ABSTRACT: Although it is widely accepted that chronic exposure to arsenite, nickel, chromium and cadmium increases cancer incidence in individuals, the molecular mechanisms underlying their ability to transform cells remain largely unknown. Carcinogenic metals are typically weak mutagens, suggesting that genetic-based mechanisms may not be primarily responsible for metal-induced carcinogenesis. Growing evidence shows that environmental metal exposure involves changes in epigenetic marks, which may lead to a possible link between heritable changes in gene expression and disease susceptibility and development. Here, we review recent advances in the understanding of metal exposure affecting epigenetic marks and discuss establishment of heritable gene expression in metal-induced carcinogenesis.
Epigenetics: official journal of the DNA Methylation Society 07/2011; 6(7):820-7. DOI:10.4161/epi.6.7.16250 · 4.78 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Homeobox genes encode transcription factors which function in body axis patterning in the developing embryo. Recent evidence suggests that the maintenance of specific HOX expression patterns is necessary for regulating the homeostasis of adult tissues as well. In this study, HOXB7 transformed human mammary epithelial cells, MCF10A, to grow in minimally supplemented medium, to form colonies in Matrigel, and display resistance to ionizing radiation. Searching for protein partners of HOXB7 that might contribute to resistance to ionizing radiation, we identified four HOXB7-binding proteins by GST pull-down/affinity chromatography and confirmed their interactions by coimmunoprecipitation in vivo. Interestingly, all four HOXB7-binding proteins shared functions as genomic caretakers and included members of the DNA-dependent protein kinase holoenzyme (Ku70, Ku80, DNA-PK(cs)) responsible for DNA double-strand break repair by nonhomologous end joining pathway and poly(ADP) ribose polymerase. Exogenous and endogenous expression of HOXB7 enhanced nonhomologous end joining and DNA repair functions in vitro and in vivo, which were reversed by silencing HOXB7. This is the first mechanistic study providing definitive evidence for the involvement of any HOX protein in DNA double-strand break repair.
Cancer Research 03/2007; 67(4):1527-35. DOI:10.1158/0008-5472.CAN-06-4283 · 9.33 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The pathophysiology of Huntington's disease reflects actions of mutant Huntingtin (Htt) (mHtt) protein with polyglutamine repeats, whose N-terminal fragment translocates to the nucleus to elicit neurotoxicity. We establish that the nuclear translocation and associated cytotoxicity of mHtt reflect a ternary complex of mHtt with GAPDH and Siah1, a ubiquitin-E3-ligase. Overexpression of GAPDH or Siah1 enhances nuclear translocation of mHtt and cytotoxicity, whereas GAPDH mutants that cannot bind Siah1 prevent translocation. Depletion of GAPDH or Siah1 by RNA interference diminishes nuclear translocation of mHtt.
Proceedings of the National Academy of Sciences 03/2006; 103(9):3405-9. DOI:10.1073/pnas.0511316103 · 9.67 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Poly(ADP-ribose) polymerase 1 (PARP-1) activity is detected in both neuronal and nonneuronal cells in the CNS, and excessive PARP-1 activity is known to be detrimental to tissue because of the cellular energy loss. Accordingly, PARP-1-deficient (PARP-1(-/-)) mice have been shown to be resistant to cerebral ischemia and several forms of inflammation. Recently, PARP-1 in glial cells has been shown to mediate the expression of proinflammatory genes in response to inflammatory stimuli by, in part, enhancing cognate DNA-binding capacities of transcription factors such as NF-kappaB and activator protein 1. Here, we demonstrate an additional mechanism whereby a significant reduction of proinflammatory gene expression such as IL-1beta, tumor necrosis factor alpha, and inducible nitricoxide synthase in PARP-1(-/-) glial cells is linked to defective inflammatory stimuli-induced p38MAPK-mediated phosphorylation of ATF-2 and cAMP-response element-binding protein and phosphorylation of NF-kappaB p65. Importantly, an inflammatory stimuli-induced p38MAPK activation is impaired in PARP-1(-/-) glial cells in a signaling pathway- and cell/tissue type-specific manner. These findings indicate that PARP-1 is an essential host factor among factors that actively mediate excessive production of proinflammatory molecules in glial cells, which may in turn contribute to the initiation of neuronal injuries.
Proceedings of the National Academy of Sciences 05/2004; 101(14):5087-92. DOI:10.1073/pnas.0306895101 · 9.67 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Poly(ADP-ribose) polymerase-1 (PARP-1, EC ), a nuclear enzyme activated by DNA strand breaks, physiologically participates in DNA repair. Excessive activation of PARP-1 by cellular insults depletes its substrate beta-nicotinamide adenine dinucleotide and ATP, leading to cell death. PARP-1-deficient (PARP-1-/-) mice are protected from several forms of inflammation. In the present study, we demonstrate in PARP-1-/- glial cells a loss of several stress-activated transcription factors as well as decreased expression of genes for cytokines and cellular adhesion molecules. We also show that augmented expression of some of these genes is independent of PARP-1 catalytic activity. These findings indicate that PARP-1 plays a pivotal role in the initial inflammatory response by modulating transcription of inflammation-linked genes.
Proceedings of the National Academy of Sciences 04/2002; 99(5):3270-5. DOI:10.1073/pnas.052712399 · 9.67 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Poly(ADP-ribose) polymerase-1 (PARP-1; EC ) is an abundant nuclear enzyme, activated by DNA strand breaks to attach up to 200 ADP-ribose groups to nuclear proteins. As retroviral infection requires integrase-catalyzed DNA strand breaks, we examined infection of pseudotyped HIV type I in fibroblasts from mice with a targeted deletion of PARP-1. Viral infection is almost totally abolished in PARP-1 knockout fibroblasts. This protection from infection reflects prevention of viral integration into the host genome. These findings suggest a potential for PARP inhibitors in therapy of HIV type I infection.
Proceedings of the National Academy of Sciences 04/2001; 98(6):3364-8. DOI:10.1073/pnas.051633498 · 9.67 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: FKBP12, the 12-kDa FK506-binding protein, is a ubiquitous abundant protein that acts as a receptor for the immunosuppressant drug FK506, binds tightly to intracellular calcium release channels and to the transforming growth factor beta (TGF-beta) type I receptor. We now demonstrate that cells from FKBP12-deficient (FKBP12(-/-)) mice manifest cell cycle arrest in G(1) phase and that these cells can be rescued by FKBP12 transfection. This arrest is mediated by marked augmentation of p21(WAF1/CIP1) levels, which cannot be further augmented by TGF-beta1. The p21 up-regulation and cell cycle arrest derive from the overactivity of TGF-beta receptor signaling, which is normally inhibited by FKBP12. Cell cycle arrest is prevented by transfection with a dominant-negative TGF-beta receptor construct. TGF-beta receptor signaling to gene expression can be mediated by SMAD, p38, and ERK/MAP kinase (extracellular signal-regulated kinase/mitogen-activated protein kinase) pathways. SMAD signaling is down-regulated in FKBP12(-/-) cells. Inhibition of ERK/MAP kinase fails to affect p21 up-regulation. By contrast, activated phosphorylated p38 is markedly augmented in FKBP12(-/-) cells and the p21 up-regulation is prevented by an inhibitor of p38. Thus, FKBP12 is a physiologic regulator of cell cycle acting by normally down-regulating TGF-beta receptor signaling.
Proceedings of the National Academy of Sciences 03/2001; 98(5):2425-30. DOI:10.1073/pnas.041614198 · 9.67 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Reactive oxygen species have recently been demonstrated to play a role in numerous cellular signal transduction pathways. Here we investigate the involvement of H2O2 in Raf-1-mediated differentiation in the human medullary thyroid carcinoma (MTC) cell line TT:deltaRaf-1:ER. Catalase, but not Cu/Zn superoxide dismutase, completely inhibited Raf-1-induced differentiation of beta-estradiol-treated TT: deltaRaf-1:ER. In addition, catalase treatment down-regulated RET expression at both the mRNA and protein levels and induced apoptosis in the parental TT cell line and uninduced TT:deltaRaf-1:ER human MTC cells. These results implicate H2O2 as a downstream mediator of c-Raf-1-induced differentiation and as a survival factor in MTC cells.
Clinical Cancer Research 10/2000; 6(9):3783-7. · 8.72 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Poly(ADP-ribose) polymerase-1 (PARP-1) is a nuclear enzyme, activated by DNA strand breaks to participate in DNA repair. Overactivation of PARP by cellular insults depletes its substrate NAD+ and then ATP, leading to a major energy deficit and cell death. This mechanism appears to be prominent in vascular stroke and other neurodegenerative processes in which PARP gene deletion and PARP-inhibiting drugs provide major protection. Cell death associated with PARP-1 overactivation appears to be predominantly necrotic while apoptosis is associated with PARP-1 cleavage, which may conserve energy needed for the apoptotic process. Novel forms of PARP derived from distinct genes and lacking classic DNA-binding domains may have nonnuclear functions, perhaps linked to cellular energy dynamics.
Neurobiology of Disease 09/2000; 7(4-7):225-239. DOI:10.1006/nbdi.2000.0324 · 5.08 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Apoptotic and necrotic cell death are well characterized and are influenced by intracellular ATP levels. Poly(ADP-ribose) polymerase (PARP), a nuclear enzyme activated by DNA strand breaks, physiologically participates in DNA repair. Overactivation of PARP after cellular insults can lead to cell death caused by depletion of the enzyme's substrate beta-nicotinamide adenine dinucleotide and of ATP. In this study, we have differentially elicited apoptosis or necrosis in mouse fibroblasts. Fibroblasts from PARP-deficient (PARP(-/-)) mice are protected from necrotic cell death and ATP depletion but not from apoptotic death. These findings, together with cell death patterns in PARP(-/-) animals receiving other types of insults, indicate that PARP activation is an active trigger of necrosis, whereas other mechanisms mediate apoptosis.
Proceedings of the National Academy of Sciences 12/1999; 96(24):13978-82. DOI:10.1073/pnas.96.24.13978 · 9.67 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Polyamine analogues such as bis(ethyl)norspermine and N1-(cyclopropylmethyl)-N11-ethyl-4,8-diazaundecane (CPENSpm) act as potent modulators of cellular polyamine metabolism in vitro and possess impressive antitumor activity against a number of cell lines. Some of these polyamine analogues appear to produce their cell-type-specific cytotoxic activity through the superinduction of spermidine/spermine N1-acetyltransferase (SSAT). However, there are several analogues (e.g., N1-(cycloheptylmethyl)-N11-ethyl-4, 8-diazaundecane (CHENSpm)) which are effective cytotoxic agents but do not superinduce SSAT. We have previously demonstrated that CPENSpm and CHENSpm both initiate the cell death program, although by different mechanisms, and that CHENSpm (but not CPENSpm) induces a G2/M cell cycle arrest. We now report that one potential mechanism by which some polyamine analogues can retard growth and ultimately produce cytotoxicity is through interference with normal tubulin polymerization. In these studies, we compare the effects of the polyamine analogues CHENSpm, CPENSpm, and (S)-N1-(2-methyl-1-butyl)-N11-ethyl-4,8-diazaundecane (IPENSpm) on in vitro tubulin polymerization. These spermine analogues behave very differently from spermine and from each other in terms of tubulin polymerization rate, equilibrium levels, and time of polymerization initiation. These results demonstrate that structurally similar polyamine analogues with potent antitumor effects can produce significantly different cellular effects. The discovery of polyamine analogues that can alter tubulin polymerization provides a series of promising lead compounds that may have a similar spectrum of activity to more difficult to synthesize compounds typified by paclitaxel.
[Show abstract][Hide abstract] ABSTRACT: The polyamines are small organic cations that are absolutely required for eukaryotic cell growth. Although their growth requirements are well established, the molecular functions of the polyamines are ill-defined. Oxidative damage to DNA by reactive oxygen species is a continual problem that cells must guard against to survive. The polyamine spermine, which is normally found in millimolar concentrations in the nucleus, is shown here to function directly as a free radical scavenger, and adducts formed as a result of this function are identified. These data suggest that spermine is a major natural intracellular compound capable of protecting DNA from free radical attack.
Proceedings of the National Academy of Sciences 10/1998; 95(19):11140-5. DOI:10.1073/pnas.95.19.11140 · 9.67 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The polyamine analogue, N1-ethyl-N11-[(cycloheptyl)methyl]-4,8-diazaundecane (CHENSpm)-induced programmed cell death in NCI H157 cells is accompanied by cytochrome c release, the loss of mitochondrial membrane potential, activation of caspase-3, caspase-mediated poly(ADP-ribose) polymerase cleavage, G2-M arrest, and DNA and nuclear fragmentation. Overexpression of Bcl-2 completely inhibits CHENSpm-induced cytochrome c release, caspase-3 activation, and poly(ADP-ribose) polymerase cleavage. However, Bcl-2 does not abrogate CHENSpm-induced programmed cell death. These results suggest that although cytochrome c release and activation of the caspase-3 protease cascade contribute to the rapid and efficient execution of apoptosis, a caspase cascade-independent pathway also exists and can be activated by CHENSpm treatment.
Cancer Research 08/1998; 58(13):2711-4. · 9.33 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Reactive oxygen species are known to induce strand breaks and/or base modifications in DNA. DNA strand breaks are associated with many pathologies and programmed cell death. We have examined the ability of the polyamines and their analogues to protect phi X-174 plasmid DNA from strand breakage induced by a oxygen-radical generating system. Spermine and several unsymmetrically substituted polyamine analogues reduced the amount of strand breakage at a physiologically relevant concentration of 1 mM. However, putrescine, spermidine, N1-acetylspermine, N1-acetylspermidine and symmetrically alkylated polyamine analogues were not able to reduce strand breakage at the same concentration. Thus, the unsymmetrically alkylated polyamine analogues and natural spermine can protect DNA against strand breakage induced by Cu(II)/H2O2 generated ROS similar to other more classical antioxidants.
Biochemical and Biophysical Research Communications 03/1998; 244(1):298-303. DOI:10.1006/bbrc.1998.8258 · 2.30 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: N1-ethyl-N11-[(cyclopropyl)methyl]-4,8,-diazaundecane (CPENSpm) is a polyamine analogue that represents a new class of antitumor agents that demonstrate phenotype-specific cytotoxic activity. However, the precise mechanism of its selective cytotoxic activity is not known. CPENSpm treatment results in the superinduction of the polyamine catabolic enzyme spermidine/spermine N1-acetyltransferase (SSAT) in sensitive cell types and has been demonstrated to induce programmed cell death (PCD). The catalysis of polyamines by the SSAT/polyamine oxidase (PAO) pathway produces H2O2 as one product, suggesting that PCD produced by CPENSpm may be, in part, due to oxidative stress as a result of H2O2 production. In the sensitive human nonsmall cell line H157, the coaddition of catalase significantly reduces high molecular weight (HMW) DNA (>/=50 kb) and nuclear fragmentation. Important to note, specific inhibition of PAO by N,N'-bis(2, 3-butadienyl)-1,4-butane-diamine results in a significant reduction of the formation of HMW DNA and nuclear fragmentation. In contrast, the coaddition of catalase or PAO inhibitor has no effect on reducing HMW DNA fragmentation induced by N1-ethyl-N11-[(cycloheptyl)methyl]-4,8,-diazaundecane, which does not induce SSAT and does not deplete intracellular polyamines. These results strongly suggest that H2O2 production by PAO has a role in CPENSpm cytotoxicity in sensitive cells via PCD and demonstrate a potential basis for differential sensitivity to this promising new class of antineoplastic agents. Furthermore, the data suggest a general mechanism by which, under certain stimuli, cells can commit suicide through catabolism of the ubiquitous intracellular polyamines.
Proceedings of the National Academy of Sciences 10/1997; 94(21):11557-62. DOI:10.1073/pnas.94.21.11557 · 9.67 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: In an effort to study the mechanism underlying the observed phenotype-specific response of human lung cancer cell lines to a polyamine analogue, N1,N12-bis(ethyl)spermine(BESpm), we have isolated a BESpm resistant cell line from the BESpm-sensitive large cell lung carcinoma line NCI H157. The mutant line exhibits identical growth rates in the presence or absence of the analogue. However, the overall growth of mutant cells reaches stationary phase earlier than that of the parental cells. In contrast to the parental cells, where a superinduction of spermidine/spermine N1-acetyltransferase (SSAT) is associated with BESpm toxicity, treatment of this resistant line with BESpm did not induce SSAT mRNA or enzyme activity. BESpm treatment was not effective in depleting the intracellular polyamine pools and very low intracellular BESpm levels were detected. This BESpm resistance is not mediated by multidrug resistance (MDR) protein, since these cells maintain their sensitivity to the antineoplastic agent adriamycin. Treatment of these cells with methylglyoxal bis(guanylhydrazone) (MGBG), an AdoMetDC inhibitor which enters cell using polyamine transport system, shows no inhibition of cell growth. Our data suggest that these mutant cells are deficient in polyamine transport. Consistent with this hypothesis, exogenous polyamines did not prevent difluoromethylornithine (DFMO) induced growth inhibition in the mutant cells.