Antifreeze proteins (AFPs) provide survival mechanism for species living in subzero environments ... more Antifreeze proteins (AFPs) provide survival mechanism for species living in subzero environments by lowering the freezing points of their body fluids effectively. The mechanism is attributed to AFPs' ability to inhibit the growth of seed ice crystals through adsorption on specific ice surfaces. We have applied dynamic REDOR (Rotational Echo Double Resonance) solid state NMR to study the threonine (Thr) side chain conformational population distribution of a site-specific Thr 13 C g and 15 N doubly labeled type I AFP in frozen aqueous solution. It is known that the Thr side chains together with those of the 4th and 8th Alanine (Ala) residues commencing from the Thrs (the 1st) in the four 11-residue repeat units form the peptide ice-binding surface. The conformational information can provide structural insight with regard to how the AFP side chains structurally interact with the ice surface. w-squared statistical analysis of the experimental REDOR data in fitting the theoretically calculated dynamic REDOR fraction curves indicates that when the AFP interacted with the ice surface in the frozen AFP solution, the conformations of the Thr side chains changed from the anti-conformations, as in the AFP crystal structure, to partial population in the anti-conformation and partial population in the two gauche conformations. This change together with the structural analysis indicates that the simultaneous interactions of the methyl groups and the hydroxyl groups of the Thr side chains with the ice surface could be the reason for the conformational population change. The analysis of the theoretical dynamic REDOR fraction curves shows that the set of experimental REDOR data may fit a number of theoretical curves with different population distributions. Thus, other structural information is needed to assist in determining the conformational population distribution of the Thr side chains.
Her-2/neu (ErbB2) oncogene, the second member of the epidermal growth factor receptor (EGFR) fami... more Her-2/neu (ErbB2) oncogene, the second member of the epidermal growth factor receptor (EGFR) family, encodes a transmembrane tyrosine kinase receptor in Her-2-positive tumors. Accumulating evidences demonstrate that signaling networks activated by EGFR and transcription factor NF-jB are associated with cell response to ionizing radiation (IR). The present study shows that overexpression of ErbB2 enhanced NF-jB activation induced by IR in human breast carcinoma MCF-7 cells transfected with ErbB2 genes (MCF-7/ErbB2). Stable transfection of dominantnegative mutant IjB (MCF-7/ErbB2/mIjB) or treatment with anti-ErbB2 antibody, Herceptin, inhibited NF-jB activation and radiosensitized MCF-7/ErbB2 cells. Consistent with NF-jB regulation, basal and IR-induced Akt, a kinase downstream of ErbB2, was activated in MCF-7/ ErbB2 cells and inhibited by Herceptin. To identify specific genes affected by ErbB2-mediated NF-jB activation, a group of IR-responsive elements Cyclin B1, Cyclin D1, Bcl-2, Bcl/XL, BAD and BAX were evaluated. Basal levels of prosurvival elements Cyclin B1, Cyclin D1, Bcl-2 and Bcl/XL but not apoptotic BAD and BAX were upregulated in MCF-7/ErbB2 cells with striking enhancements in Bcl-2 and Bcl/XL. IR further induced Cyclin B1 and Cyclin D1 expression that was reduced by Herceptin. Bcl-2 kept a high steady level after Herceptin þ IR treatment and, in contrast to control MCF-7/Vector cells, Bcl/XL was inhibited in MCF-7/ErbB2 cells by Herceptin þ IR treatment. However, all four prosurvival proteins were downregulated by inhibition of NF-jB in MCF-7/ErbB2/mIjB cells. These results thus provide evidence suggesting that overexpression of ErbB2 is able to enhance NF-jB response to IR, and that a specific prosurvival network downstream of NF-jB is triggered by treatments using anti-ErbB2 antibody combined with radiation.
Glutathione S-transferase P1–1 (GSTp) is an abundant and ubiq-uitously expressed protein in norma... more Glutathione S-transferase P1–1 (GSTp) is an abundant and ubiq-uitously expressed protein in normal and malignant mammalian tissues and possesses catalytic and ligand binding properties. Our present data suggest that the protein contributes to the regulation of cell proliferation. Mouse embryo fibroblasts (MEFs) isolated from mice with a GSTP1–1 [glutathione S-transferase P1–1 (isozyme in nonhepatic tissue)] null genotype (GSTp2/2) doubled their population in 26.2 h versus 33.6 h for the wild type (GSTp1/1). Retroviral transfection of GSTP1–1 into GSTp2/2 MEF cells slowed the doubling time to 30.4 h. Both early passage and immortalized MEF cells from GSTp2/2 animals expressed signif-icantly elevated activity of extracellular signal-regulated kinases ERK1/ERK2, kinases linked to cell proliferation pathways. In vivo, GSTp2/2 mice had higher basal levels of circulating white blood
Phospholipase C (PLC)-b1 and PLC-b2 are regulated by the Gq family of heterotrimeric G proteins a... more Phospholipase C (PLC)-b1 and PLC-b2 are regulated by the Gq family of heterotrimeric G proteins and contain C2 domains. These domains are Ca21-binding modules that serve as membrane-attachment motifs in a number of signal transduction proteins. To determine the role that C2 domains play in PLC-b1 and PLC-b2 function, we measured the binding of the isolated C2 domains to membrane bilayers. We found, unexpectedly, that these modules do not bind to membranes but they associate strongly and specifically to activated [guanosine 5*-[g-thio]triphosphate (GTP[gS])bound] Gaq subunits. The C2 domain of PLC-b1 effectively suppressed the activation of the intact isozyme by Gaq(GTP[gS]), indicating that the C2-Gaq interaction may be physiologically relevant. C2 affinity for Gaq(GTP[gS]) was reduced when Gaq was deactivated to the GDP-bound state. Binding to activated Gai1 subunits or to Gbg subunits was not detected. Also, Gaq(GTP[gS]) failed to associate with the C2 domain of PLC-d, an isozyme ...
Nuclear factor-kappaB (NF-kappaB) is one of the key regulatory molecules in oxidative stress-indu... more Nuclear factor-kappaB (NF-kappaB) is one of the key regulatory molecules in oxidative stress-induced cell activation. NF-kappaB is normally sequestered in the cytoplasm of nonstimulated cells and must translocate into the nucleus to regulate effector gene expression. A family of inhibitory proteins, IKBs, binds to NF-kappaB and masks its nuclear localization signal domain and therefore controls the translocation of NF-kappaB. Exposure of cells to extracellular stimuli that perturb redox balance results in rapid phosphorylation, ubiquitination, and proteolytic degradation of IkappaBs. This process frees NF-kappaB from the NF-KB/IKB complexes and enables NF-kappaB to translocate to the nucleus where it regulates gene transcription. Many effector genes including those encoding cytokines and adhesion molecules are in turn regulated by NF-kappaB. NF-kappaB is also an essential component of ionizing radiation (IR)-triggered signal transduction pathways that can lead to cell death or survi...
BACKGROUND To understand the molecular response of tumor cells to therapeutic ionizing radiation ... more BACKGROUND To understand the molecular response of tumor cells to therapeutic ionizing radiation (IR), we previously reported that human breast cancer cells derived following chronic exposure to fractionated ionizing radiation (MCF+FIR) showed a transient radioresistance. MCF+FIR cells also demonstrated increased activity of NF-kappaB, increased expression of the mitochondrial antioxidant enzyme (MnSOD), and increased expression of a cell cycle regulatory protein (Cyclin B1). The present studies were designed to determine the relationship of NF-kappaB, MnSOD and Cyclin B1 expression in cellular adaptive responses to ionizing radiation. MATERIALS AND METHODS The first intron of the cyclin B1 gene with a putative NF-kappaB element was cloned into the pGL3 luciferase reporter (pGL3CB1EI1). PGL3CB1EI1 and control NF-kappaB luciferase activities were determined in MCF-7 and MCF+FIR cells treated with a single dose of radiation, over expression of the dominant negative mutant IkB (mIkB) o...
Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA In our previous study, we i... more Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA In our previous study, we identified a protein target, Peroxiredoxin II (PrxII) to be overexpressed in radioresistant MCF+FIR3 breast cancer cells and found that its expression and function is associated with breast cancer radiation sensitivity or resistance. Small interference RNA (siRNA) targeting the PrxII gene expression was able to sensitize MCF+FIR3 radioresistant breast cancer cells to ionizing radiation. The major focus of this work was to investigate how the radiation response of MCF+FIR3 radioresistant cells was affected by siRNA that inhibits PrxII gene expression. Our results presented here show that silencing PrxII gene expression increased cellular toxicity by altering cellular thiol status, inhibiting Ca2+ efflux from the cells and perturbing the intracellular Ca2+ homeostasis. By combining radiotherapy and siRNA technology, we hope to develop new therapeutic strategies that may have potential to enhance the efficacy of chemotherapeutic agents due to its targeted property to specific cancer related genes. Citation Format: Tieli Wang, Anthony Joseph Diaz, Jian-Jian Li, Yun Yen, Daniel Tamae. Enhanced radiation reponse in MCF-7 radioresistant cells by targeting Peroxiredoxin II. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3953. doi:10.1158/1538-7445.AM2014-3953
The alkylating agent, temozolomide (TMZ), is considered the standard-of-care for high-grade astro... more The alkylating agent, temozolomide (TMZ), is considered the standard-of-care for high-grade astrocytomas -known as glioblastoma multiforme (GBM)- an aggressive type of tumor with poor prognosis. The therapeutic benefit of TMZ is attributed to formation of DNA adducts involving the methylation of purine bases in DNA. We investigated the effects of TMZ on arginine and lysine amino acids, histone H3 peptides and histone H3 proteins. Chemical modification of amino acids, histone H3 peptide and protein by TMZ was performed in phosphate buffer at physiological pH. The reaction products were examined by mass spectrometry and western blot analysis. Our results showed that TMZ following conversion to a methylating cation, can methylate histone H3 peptide and histone H3 protein, suggesting that TMZ exerts its anticancer activity not only through its interaction with DNA, but also through alterations of protein post-translational modifications. The possibility that TMZ can methylate histones i...
Pleckstrin homology (PH) domains are recognized in more than 100 different proteins, including ma... more Pleckstrin homology (PH) domains are recognized in more than 100 different proteins, including mammalian phosphoinositide-specific phospholipase C (PLC) isozymes (isotypes , γ, and δ). These structural motifs are thought to function as tethering devices linking their host proteins to membranes containing phosphoinositides or γ subunits of heterotrimeric GTP binding (G) proteins. Although the PH domains of PLC-δ and PLC-γ have been studied, the comparable domains of the isotypes have not. Here, we have measured the affinities of the isolated PH domains of PLC-1 and-2 (PH-1 and PH-2 , respectively) for lipid bilayers and G-γ subunits. Like the intact enzymes, these PH domains bind to membrane surfaces composed of zwitterionic phosphatidylcholine with moderate affinity. Inclusion of the anionic lipid phosphatidylserine or phosphatidylinositol 4,5-bisphosphate [PI(4,5)P 2 ] and inclusion of G-γ subunits had little affect on their membrane affinity. In contrast, binding of PLC-δ 1 or its PH domain was highly dependent on PI(4,5)P 2. We also determined whether these domains laterally associate with G-γ subunits bound to membrane surfaces using fluorescence resonance energy transfer. Affinities for G-γ were in the following order: PH-2 g PH-1 > PH-δ 1 ; the affinities of the native enzyme were as follows: PLC-2. PLC-δ 1 > PLC-1. Thus, the PH domain of PLC-1 interacts with G-γ in isolation, but not in the context of the native enzyme. By contrast, docking of the PH domain of PLC-2 with G-γ is comparable to that of the full-length protein and may play a key role in G-γ recognition.
Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA In our previous study, we i... more Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA In our previous study, we identified a protein target, Peroxiredoxin II (PrxII) to be overexpressed in radioresistant MCF+FIR3 breast cancer cells and found that its expression and function is associated with breast cancer radiation sensitivity or resistance. Small interference RNA (siRNA) targeting the PrxII gene expression was able to sensitize MCF+FIR3 radioresistant breast cancer cells to ionizing radiation. The major focus of this work was to investigate how the radiation response of MCF+FIR3 radioresistant cells was affected by siRNA that inhibits PrxII gene expression. Our results presented here show that silencing PrxII gene expression increased cellular toxicity by altering cellular thiol status, inhibiting Ca2+ efflux from the cells and perturbing the intracellular Ca2+ homeostasis. By combining radiotherapy and siRNA technology, we hope to develop new therapeutic strategies that may have potential to enhance the efficacy of chemotherapeutic agents due to its targeted property to specific cancer related genes. Citation Format: Tieli Wang, Anthony Joseph Diaz, Jian-Jian Li, Yun Yen, Daniel Tamae. Enhanced radiation reponse in MCF-7 radioresistant cells by targeting Peroxiredoxin II. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3953. doi:10.1158/1538-7445.AM2014-3953
Journal of Pharmacology and Experimental Therapeutics
Glutathione S-transferase P1-1 (GSTpi) is an abundant and ubiquitously expressed protein in norma... more Glutathione S-transferase P1-1 (GSTpi) is an abundant and ubiquitously expressed protein in normal and malignant mammalian tissues and possesses catalytic and ligand binding properties. Our present data suggest that the protein contributes to the regulation of cell proliferation. Mouse embryo fibroblasts (MEFs) isolated from mice with a GSTP1-1 [glutathione S-transferase P1-1 (isozyme in nonhepatic tissue)] null genotype (GSTpi(-/-)) doubled their population in 26.2 h versus 33.6 h for the wild type (GSTpi(+/+)). Retroviral transfection of GSTP1-1 into GSTpi(-/-) MEF cells slowed the doubling time to 30.4 h. Both early passage and immortalized MEF cells from GSTpi(-/-) animals expressed significantly elevated activity of extracellular signal-regulated kinases ERK1/ERK2, kinases linked to cell proliferation pathways. In vivo, GSTpi(-/-) mice had higher basal levels of circulating white blood cells compared with GSTpi(+/+). Administration of a peptidomimetic inhibitor of GSTP1-1, TLK199, (gamma-glutamyl-S-(benzyl)cysteinyl-R-phenyl glycine diethyl ester), stimulated both lymphocyte production and bone marrow progenitor (colony-forming unit-granulocyte macrophage) proliferation, but only in GSTpi(+/+) and not in GSTpi(-/-) animals. Selection of a resistant clone of an HL60 tumor cell line through chronic exposure to TLK199 resulted in cells with elevated activities of c-Jun NH2 terminal kinase (JNK1) and ERK1/ERK2, and allowed the cells to proliferate under stress conditions that induced high levels of apoptosis in the wild type cells. The in vitro and in vivo data are consistent with the principle that GSTP1-1 influences cell proliferation.
To understand the molecular response of tumor cells to therapeutic ionizing radiation (IR), we pr... more To understand the molecular response of tumor cells to therapeutic ionizing radiation (IR), we previously reported that human breast cancer cells derived following chronic exposure to fractionated ionizing radiation (MCF+FIR) showed a transient radioresistance. MCF+FIR cells also demonstrated increased activity of NF-kappaB, increased expression of the mitochondrial antioxidant enzyme (MnSOD), and increased expression of a cell cycle regulatory protein (Cyclin B1). The present studies were designed to determine the relationship of NF-kappaB, MnSOD and Cyclin B1 expression in cellular adaptive responses to ionizing radiation. The first intron of the cyclin B1 gene with a putative NF-kappaB element was cloned into the pGL3 luciferase reporter (pGL3CB1EI1). PGL3CB1EI1 and control NF-kappaB luciferase activities were determined in MCF-7 and MCF+FIR cells treated with a single dose of radiation, over expression of the dominant negative mutant IkB (mIkB) or over expression of the SOD2 gen...
International journal of biological sciences, Jan 4, 2007
Epidemiological data have suggested an increased cancer rates in diabetic patients, for which the... more Epidemiological data have suggested an increased cancer rates in diabetic patients, for which the underlying mechanism is poorly understood. We studied whether high level of glucose (HG) treatment that mimic the hyperglycemic condition in diabetes mellitus is mutagenic. Mutagenesis studies were carried out at both hypoxanthine phosphoribosyltransferase (hprt) and thymidine kinase (tk) loci. Role of p53 in HG-induced mutagenesis was also investigated by using human lymphoblastoid cell lines derived from same donor but differs in p53 statuses; TK6 has wild-type p53, NH32 has null p53, and WTK1 has mutant p53 (ile237). In addition, we studied the influence of antioxidant treatment on HG-induced mutagenesis. Mutation fractions at both loci increased significantly in all three lines at 21 and 28 days after HG treatments. At tk locus, the increase of a class of mutants with normal growth rate is mainly responsible for the overall increased mutant fraction. Compared to TK6 cells, both NH32...
Peroxiredoxin (Prx)II belongs to a family of redox-active proteins that use redox-sensitive cyste... more Peroxiredoxin (Prx)II belongs to a family of redox-active proteins that use redox-sensitive cysteine in the active site to reduce peroxides. PrxII is induced by various oxidative stimuli and plays an important protective role against oxidative radical damage by reactive oxygen species. PrxII expression levels are correlated with resistance to radiation therapy or certain anti-cancer drugs in radioresistant breast cancer cells, glioblastomas, and head and neck cancer cells as well as in tissue isolated from head and neck patients who do not respond to radiation therapy. Small interfering RNA (siRNA) that inhibits the PrxII gene expression has been shown to partially reverse the radioresistant phenotype in radiation resistant breast cancer cells and sensitizes glioma cells to oxidative stress, highlighting the potential clinical importance of PrxII in radiation resistance in cancer. This article focuses on the role that PrxII may play in chemoresistant breast cancer cells.
... 2291 Syntheses and Reactions of Iron and Ruthenium Complexes of an Optically Pure Fused Cyclo... more ... 2291 Syntheses and Reactions of Iron and Ruthenium Complexes of an Optically Pure Fused Cyclopentadienyl Ligand Debjani Bhaduri and John H. Nelson* ... Chem. Soc. 1983,105,679. (4) Morandini, F.; Consiglio, G.; Lucchini, V. Organometallics 1985, 4, 1202. ...
Page 1. 4474 Organometallics 1994, 13, 4474-4480 Synthesis, Equilibrium Binding, and 77Se NMR Stu... more Page 1. 4474 Organometallics 1994, 13, 4474-4480 Synthesis, Equilibrium Binding, and 77Se NMR Studies of gl-Selenophene (Seln) Complexes: [CpRu(CO)(PPhs)(g1(Se)-Seln)]BF4 Carter J. White, Tieli Wang, R. A. Jacobson, and Robert J. Angelici* ...
Antioxidant enzymes are critical in oxidative stress responses. Radioresistant variants isolated ... more Antioxidant enzymes are critical in oxidative stress responses. Radioresistant variants isolated from MCF-7 human carcinoma cells following fractionated ionizing radiation (MCF+FIR cells) or overexpression of manganese superoxide dismutase (MCF+SOD cells) demonstrated dose-modifying factors at 10% isosurvival of 1.8 and 2.3, respectively. MCF+FIR and MCF-7 cells (exposed to single-dose radiation) demonstrated 5- to 10-fold increases in MnSOD activity, mRNA, and immunoreactive protein. Radioresistance in MCF+FIR and MCF+SOD cells was reduced following expression of antisense MnSOD. DNA microarray analysis and immunoblotting identified p21, Myc, 14-3-3 zeta, cyclin A, cyclin B1, and GADD153 as genes constitutively overexpressed (2- to 10-fold) in both MCF+FIR and MCF+SOD cells. Radiation-induced expression of these six genes was suppressed in fibroblasts from Sod2 knockout mice (−/−) as well as in MCF+FIR and MCF+SOD cells expressing antisense MnSOD. Inhibiting NF-κB transcriptional a...
Antifreeze proteins (AFPs) provide survival mechanism for species living in subzero environments ... more Antifreeze proteins (AFPs) provide survival mechanism for species living in subzero environments by lowering the freezing points of their body fluids effectively. The mechanism is attributed to AFPs' ability to inhibit the growth of seed ice crystals through adsorption on specific ice surfaces. We have applied dynamic REDOR (Rotational Echo Double Resonance) solid state NMR to study the threonine (Thr) side chain conformational population distribution of a site-specific Thr 13 C g and 15 N doubly labeled type I AFP in frozen aqueous solution. It is known that the Thr side chains together with those of the 4th and 8th Alanine (Ala) residues commencing from the Thrs (the 1st) in the four 11-residue repeat units form the peptide ice-binding surface. The conformational information can provide structural insight with regard to how the AFP side chains structurally interact with the ice surface. w-squared statistical analysis of the experimental REDOR data in fitting the theoretically calculated dynamic REDOR fraction curves indicates that when the AFP interacted with the ice surface in the frozen AFP solution, the conformations of the Thr side chains changed from the anti-conformations, as in the AFP crystal structure, to partial population in the anti-conformation and partial population in the two gauche conformations. This change together with the structural analysis indicates that the simultaneous interactions of the methyl groups and the hydroxyl groups of the Thr side chains with the ice surface could be the reason for the conformational population change. The analysis of the theoretical dynamic REDOR fraction curves shows that the set of experimental REDOR data may fit a number of theoretical curves with different population distributions. Thus, other structural information is needed to assist in determining the conformational population distribution of the Thr side chains.
Her-2/neu (ErbB2) oncogene, the second member of the epidermal growth factor receptor (EGFR) fami... more Her-2/neu (ErbB2) oncogene, the second member of the epidermal growth factor receptor (EGFR) family, encodes a transmembrane tyrosine kinase receptor in Her-2-positive tumors. Accumulating evidences demonstrate that signaling networks activated by EGFR and transcription factor NF-jB are associated with cell response to ionizing radiation (IR). The present study shows that overexpression of ErbB2 enhanced NF-jB activation induced by IR in human breast carcinoma MCF-7 cells transfected with ErbB2 genes (MCF-7/ErbB2). Stable transfection of dominantnegative mutant IjB (MCF-7/ErbB2/mIjB) or treatment with anti-ErbB2 antibody, Herceptin, inhibited NF-jB activation and radiosensitized MCF-7/ErbB2 cells. Consistent with NF-jB regulation, basal and IR-induced Akt, a kinase downstream of ErbB2, was activated in MCF-7/ ErbB2 cells and inhibited by Herceptin. To identify specific genes affected by ErbB2-mediated NF-jB activation, a group of IR-responsive elements Cyclin B1, Cyclin D1, Bcl-2, Bcl/XL, BAD and BAX were evaluated. Basal levels of prosurvival elements Cyclin B1, Cyclin D1, Bcl-2 and Bcl/XL but not apoptotic BAD and BAX were upregulated in MCF-7/ErbB2 cells with striking enhancements in Bcl-2 and Bcl/XL. IR further induced Cyclin B1 and Cyclin D1 expression that was reduced by Herceptin. Bcl-2 kept a high steady level after Herceptin þ IR treatment and, in contrast to control MCF-7/Vector cells, Bcl/XL was inhibited in MCF-7/ErbB2 cells by Herceptin þ IR treatment. However, all four prosurvival proteins were downregulated by inhibition of NF-jB in MCF-7/ErbB2/mIjB cells. These results thus provide evidence suggesting that overexpression of ErbB2 is able to enhance NF-jB response to IR, and that a specific prosurvival network downstream of NF-jB is triggered by treatments using anti-ErbB2 antibody combined with radiation.
Glutathione S-transferase P1–1 (GSTp) is an abundant and ubiq-uitously expressed protein in norma... more Glutathione S-transferase P1–1 (GSTp) is an abundant and ubiq-uitously expressed protein in normal and malignant mammalian tissues and possesses catalytic and ligand binding properties. Our present data suggest that the protein contributes to the regulation of cell proliferation. Mouse embryo fibroblasts (MEFs) isolated from mice with a GSTP1–1 [glutathione S-transferase P1–1 (isozyme in nonhepatic tissue)] null genotype (GSTp2/2) doubled their population in 26.2 h versus 33.6 h for the wild type (GSTp1/1). Retroviral transfection of GSTP1–1 into GSTp2/2 MEF cells slowed the doubling time to 30.4 h. Both early passage and immortalized MEF cells from GSTp2/2 animals expressed signif-icantly elevated activity of extracellular signal-regulated kinases ERK1/ERK2, kinases linked to cell proliferation pathways. In vivo, GSTp2/2 mice had higher basal levels of circulating white blood
Phospholipase C (PLC)-b1 and PLC-b2 are regulated by the Gq family of heterotrimeric G proteins a... more Phospholipase C (PLC)-b1 and PLC-b2 are regulated by the Gq family of heterotrimeric G proteins and contain C2 domains. These domains are Ca21-binding modules that serve as membrane-attachment motifs in a number of signal transduction proteins. To determine the role that C2 domains play in PLC-b1 and PLC-b2 function, we measured the binding of the isolated C2 domains to membrane bilayers. We found, unexpectedly, that these modules do not bind to membranes but they associate strongly and specifically to activated [guanosine 5*-[g-thio]triphosphate (GTP[gS])bound] Gaq subunits. The C2 domain of PLC-b1 effectively suppressed the activation of the intact isozyme by Gaq(GTP[gS]), indicating that the C2-Gaq interaction may be physiologically relevant. C2 affinity for Gaq(GTP[gS]) was reduced when Gaq was deactivated to the GDP-bound state. Binding to activated Gai1 subunits or to Gbg subunits was not detected. Also, Gaq(GTP[gS]) failed to associate with the C2 domain of PLC-d, an isozyme ...
Nuclear factor-kappaB (NF-kappaB) is one of the key regulatory molecules in oxidative stress-indu... more Nuclear factor-kappaB (NF-kappaB) is one of the key regulatory molecules in oxidative stress-induced cell activation. NF-kappaB is normally sequestered in the cytoplasm of nonstimulated cells and must translocate into the nucleus to regulate effector gene expression. A family of inhibitory proteins, IKBs, binds to NF-kappaB and masks its nuclear localization signal domain and therefore controls the translocation of NF-kappaB. Exposure of cells to extracellular stimuli that perturb redox balance results in rapid phosphorylation, ubiquitination, and proteolytic degradation of IkappaBs. This process frees NF-kappaB from the NF-KB/IKB complexes and enables NF-kappaB to translocate to the nucleus where it regulates gene transcription. Many effector genes including those encoding cytokines and adhesion molecules are in turn regulated by NF-kappaB. NF-kappaB is also an essential component of ionizing radiation (IR)-triggered signal transduction pathways that can lead to cell death or survi...
BACKGROUND To understand the molecular response of tumor cells to therapeutic ionizing radiation ... more BACKGROUND To understand the molecular response of tumor cells to therapeutic ionizing radiation (IR), we previously reported that human breast cancer cells derived following chronic exposure to fractionated ionizing radiation (MCF+FIR) showed a transient radioresistance. MCF+FIR cells also demonstrated increased activity of NF-kappaB, increased expression of the mitochondrial antioxidant enzyme (MnSOD), and increased expression of a cell cycle regulatory protein (Cyclin B1). The present studies were designed to determine the relationship of NF-kappaB, MnSOD and Cyclin B1 expression in cellular adaptive responses to ionizing radiation. MATERIALS AND METHODS The first intron of the cyclin B1 gene with a putative NF-kappaB element was cloned into the pGL3 luciferase reporter (pGL3CB1EI1). PGL3CB1EI1 and control NF-kappaB luciferase activities were determined in MCF-7 and MCF+FIR cells treated with a single dose of radiation, over expression of the dominant negative mutant IkB (mIkB) o...
Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA In our previous study, we i... more Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA In our previous study, we identified a protein target, Peroxiredoxin II (PrxII) to be overexpressed in radioresistant MCF+FIR3 breast cancer cells and found that its expression and function is associated with breast cancer radiation sensitivity or resistance. Small interference RNA (siRNA) targeting the PrxII gene expression was able to sensitize MCF+FIR3 radioresistant breast cancer cells to ionizing radiation. The major focus of this work was to investigate how the radiation response of MCF+FIR3 radioresistant cells was affected by siRNA that inhibits PrxII gene expression. Our results presented here show that silencing PrxII gene expression increased cellular toxicity by altering cellular thiol status, inhibiting Ca2+ efflux from the cells and perturbing the intracellular Ca2+ homeostasis. By combining radiotherapy and siRNA technology, we hope to develop new therapeutic strategies that may have potential to enhance the efficacy of chemotherapeutic agents due to its targeted property to specific cancer related genes. Citation Format: Tieli Wang, Anthony Joseph Diaz, Jian-Jian Li, Yun Yen, Daniel Tamae. Enhanced radiation reponse in MCF-7 radioresistant cells by targeting Peroxiredoxin II. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3953. doi:10.1158/1538-7445.AM2014-3953
The alkylating agent, temozolomide (TMZ), is considered the standard-of-care for high-grade astro... more The alkylating agent, temozolomide (TMZ), is considered the standard-of-care for high-grade astrocytomas -known as glioblastoma multiforme (GBM)- an aggressive type of tumor with poor prognosis. The therapeutic benefit of TMZ is attributed to formation of DNA adducts involving the methylation of purine bases in DNA. We investigated the effects of TMZ on arginine and lysine amino acids, histone H3 peptides and histone H3 proteins. Chemical modification of amino acids, histone H3 peptide and protein by TMZ was performed in phosphate buffer at physiological pH. The reaction products were examined by mass spectrometry and western blot analysis. Our results showed that TMZ following conversion to a methylating cation, can methylate histone H3 peptide and histone H3 protein, suggesting that TMZ exerts its anticancer activity not only through its interaction with DNA, but also through alterations of protein post-translational modifications. The possibility that TMZ can methylate histones i...
Pleckstrin homology (PH) domains are recognized in more than 100 different proteins, including ma... more Pleckstrin homology (PH) domains are recognized in more than 100 different proteins, including mammalian phosphoinositide-specific phospholipase C (PLC) isozymes (isotypes , γ, and δ). These structural motifs are thought to function as tethering devices linking their host proteins to membranes containing phosphoinositides or γ subunits of heterotrimeric GTP binding (G) proteins. Although the PH domains of PLC-δ and PLC-γ have been studied, the comparable domains of the isotypes have not. Here, we have measured the affinities of the isolated PH domains of PLC-1 and-2 (PH-1 and PH-2 , respectively) for lipid bilayers and G-γ subunits. Like the intact enzymes, these PH domains bind to membrane surfaces composed of zwitterionic phosphatidylcholine with moderate affinity. Inclusion of the anionic lipid phosphatidylserine or phosphatidylinositol 4,5-bisphosphate [PI(4,5)P 2 ] and inclusion of G-γ subunits had little affect on their membrane affinity. In contrast, binding of PLC-δ 1 or its PH domain was highly dependent on PI(4,5)P 2. We also determined whether these domains laterally associate with G-γ subunits bound to membrane surfaces using fluorescence resonance energy transfer. Affinities for G-γ were in the following order: PH-2 g PH-1 > PH-δ 1 ; the affinities of the native enzyme were as follows: PLC-2. PLC-δ 1 > PLC-1. Thus, the PH domain of PLC-1 interacts with G-γ in isolation, but not in the context of the native enzyme. By contrast, docking of the PH domain of PLC-2 with G-γ is comparable to that of the full-length protein and may play a key role in G-γ recognition.
Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA In our previous study, we i... more Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA In our previous study, we identified a protein target, Peroxiredoxin II (PrxII) to be overexpressed in radioresistant MCF+FIR3 breast cancer cells and found that its expression and function is associated with breast cancer radiation sensitivity or resistance. Small interference RNA (siRNA) targeting the PrxII gene expression was able to sensitize MCF+FIR3 radioresistant breast cancer cells to ionizing radiation. The major focus of this work was to investigate how the radiation response of MCF+FIR3 radioresistant cells was affected by siRNA that inhibits PrxII gene expression. Our results presented here show that silencing PrxII gene expression increased cellular toxicity by altering cellular thiol status, inhibiting Ca2+ efflux from the cells and perturbing the intracellular Ca2+ homeostasis. By combining radiotherapy and siRNA technology, we hope to develop new therapeutic strategies that may have potential to enhance the efficacy of chemotherapeutic agents due to its targeted property to specific cancer related genes. Citation Format: Tieli Wang, Anthony Joseph Diaz, Jian-Jian Li, Yun Yen, Daniel Tamae. Enhanced radiation reponse in MCF-7 radioresistant cells by targeting Peroxiredoxin II. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3953. doi:10.1158/1538-7445.AM2014-3953
Journal of Pharmacology and Experimental Therapeutics
Glutathione S-transferase P1-1 (GSTpi) is an abundant and ubiquitously expressed protein in norma... more Glutathione S-transferase P1-1 (GSTpi) is an abundant and ubiquitously expressed protein in normal and malignant mammalian tissues and possesses catalytic and ligand binding properties. Our present data suggest that the protein contributes to the regulation of cell proliferation. Mouse embryo fibroblasts (MEFs) isolated from mice with a GSTP1-1 [glutathione S-transferase P1-1 (isozyme in nonhepatic tissue)] null genotype (GSTpi(-/-)) doubled their population in 26.2 h versus 33.6 h for the wild type (GSTpi(+/+)). Retroviral transfection of GSTP1-1 into GSTpi(-/-) MEF cells slowed the doubling time to 30.4 h. Both early passage and immortalized MEF cells from GSTpi(-/-) animals expressed significantly elevated activity of extracellular signal-regulated kinases ERK1/ERK2, kinases linked to cell proliferation pathways. In vivo, GSTpi(-/-) mice had higher basal levels of circulating white blood cells compared with GSTpi(+/+). Administration of a peptidomimetic inhibitor of GSTP1-1, TLK199, (gamma-glutamyl-S-(benzyl)cysteinyl-R-phenyl glycine diethyl ester), stimulated both lymphocyte production and bone marrow progenitor (colony-forming unit-granulocyte macrophage) proliferation, but only in GSTpi(+/+) and not in GSTpi(-/-) animals. Selection of a resistant clone of an HL60 tumor cell line through chronic exposure to TLK199 resulted in cells with elevated activities of c-Jun NH2 terminal kinase (JNK1) and ERK1/ERK2, and allowed the cells to proliferate under stress conditions that induced high levels of apoptosis in the wild type cells. The in vitro and in vivo data are consistent with the principle that GSTP1-1 influences cell proliferation.
To understand the molecular response of tumor cells to therapeutic ionizing radiation (IR), we pr... more To understand the molecular response of tumor cells to therapeutic ionizing radiation (IR), we previously reported that human breast cancer cells derived following chronic exposure to fractionated ionizing radiation (MCF+FIR) showed a transient radioresistance. MCF+FIR cells also demonstrated increased activity of NF-kappaB, increased expression of the mitochondrial antioxidant enzyme (MnSOD), and increased expression of a cell cycle regulatory protein (Cyclin B1). The present studies were designed to determine the relationship of NF-kappaB, MnSOD and Cyclin B1 expression in cellular adaptive responses to ionizing radiation. The first intron of the cyclin B1 gene with a putative NF-kappaB element was cloned into the pGL3 luciferase reporter (pGL3CB1EI1). PGL3CB1EI1 and control NF-kappaB luciferase activities were determined in MCF-7 and MCF+FIR cells treated with a single dose of radiation, over expression of the dominant negative mutant IkB (mIkB) or over expression of the SOD2 gen...
International journal of biological sciences, Jan 4, 2007
Epidemiological data have suggested an increased cancer rates in diabetic patients, for which the... more Epidemiological data have suggested an increased cancer rates in diabetic patients, for which the underlying mechanism is poorly understood. We studied whether high level of glucose (HG) treatment that mimic the hyperglycemic condition in diabetes mellitus is mutagenic. Mutagenesis studies were carried out at both hypoxanthine phosphoribosyltransferase (hprt) and thymidine kinase (tk) loci. Role of p53 in HG-induced mutagenesis was also investigated by using human lymphoblastoid cell lines derived from same donor but differs in p53 statuses; TK6 has wild-type p53, NH32 has null p53, and WTK1 has mutant p53 (ile237). In addition, we studied the influence of antioxidant treatment on HG-induced mutagenesis. Mutation fractions at both loci increased significantly in all three lines at 21 and 28 days after HG treatments. At tk locus, the increase of a class of mutants with normal growth rate is mainly responsible for the overall increased mutant fraction. Compared to TK6 cells, both NH32...
Peroxiredoxin (Prx)II belongs to a family of redox-active proteins that use redox-sensitive cyste... more Peroxiredoxin (Prx)II belongs to a family of redox-active proteins that use redox-sensitive cysteine in the active site to reduce peroxides. PrxII is induced by various oxidative stimuli and plays an important protective role against oxidative radical damage by reactive oxygen species. PrxII expression levels are correlated with resistance to radiation therapy or certain anti-cancer drugs in radioresistant breast cancer cells, glioblastomas, and head and neck cancer cells as well as in tissue isolated from head and neck patients who do not respond to radiation therapy. Small interfering RNA (siRNA) that inhibits the PrxII gene expression has been shown to partially reverse the radioresistant phenotype in radiation resistant breast cancer cells and sensitizes glioma cells to oxidative stress, highlighting the potential clinical importance of PrxII in radiation resistance in cancer. This article focuses on the role that PrxII may play in chemoresistant breast cancer cells.
... 2291 Syntheses and Reactions of Iron and Ruthenium Complexes of an Optically Pure Fused Cyclo... more ... 2291 Syntheses and Reactions of Iron and Ruthenium Complexes of an Optically Pure Fused Cyclopentadienyl Ligand Debjani Bhaduri and John H. Nelson* ... Chem. Soc. 1983,105,679. (4) Morandini, F.; Consiglio, G.; Lucchini, V. Organometallics 1985, 4, 1202. ...
Page 1. 4474 Organometallics 1994, 13, 4474-4480 Synthesis, Equilibrium Binding, and 77Se NMR Stu... more Page 1. 4474 Organometallics 1994, 13, 4474-4480 Synthesis, Equilibrium Binding, and 77Se NMR Studies of gl-Selenophene (Seln) Complexes: [CpRu(CO)(PPhs)(g1(Se)-Seln)]BF4 Carter J. White, Tieli Wang, R. A. Jacobson, and Robert J. Angelici* ...
Antioxidant enzymes are critical in oxidative stress responses. Radioresistant variants isolated ... more Antioxidant enzymes are critical in oxidative stress responses. Radioresistant variants isolated from MCF-7 human carcinoma cells following fractionated ionizing radiation (MCF+FIR cells) or overexpression of manganese superoxide dismutase (MCF+SOD cells) demonstrated dose-modifying factors at 10% isosurvival of 1.8 and 2.3, respectively. MCF+FIR and MCF-7 cells (exposed to single-dose radiation) demonstrated 5- to 10-fold increases in MnSOD activity, mRNA, and immunoreactive protein. Radioresistance in MCF+FIR and MCF+SOD cells was reduced following expression of antisense MnSOD. DNA microarray analysis and immunoblotting identified p21, Myc, 14-3-3 zeta, cyclin A, cyclin B1, and GADD153 as genes constitutively overexpressed (2- to 10-fold) in both MCF+FIR and MCF+SOD cells. Radiation-induced expression of these six genes was suppressed in fibroblasts from Sod2 knockout mice (−/−) as well as in MCF+FIR and MCF+SOD cells expressing antisense MnSOD. Inhibiting NF-κB transcriptional a...
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