46 results on '"Rong-Hua Tao"'
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2. Supplemental Materials and Methods from Regulation of USP37 Expression by REST-Associated G9a-Dependent Histone Methylation
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Vidya Gopalakrishnan, Gianluca Sbardella, Pete H. Taylor, Sabrina Castellano, Ciro Milite, Rong-Hua Tao, Shavali Shaik, Chandra M. Das, Jyothishmathi Swaminathan, Rashieda J. Hatcher, and Tara H.W. Dobson
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Primers for QRT-PCR Assay, Antibodies for ChIP, Primers for qChIP Assay, Antibodies for Western Blotting and Antibodies for IHC.
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- 2023
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3. Supplementary figure legends and materials from HuR Suppresses Fas Expression and Correlates with Patient Outcome in Liver Cancer
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Felipe Samaniego, Asif Rashid, Boris Blechacz, Anita L. Sabichi, Lei Feng, Xue Ao, Rong-Hua Tao, Tamer Khashab, Lalit Sehgal, Rohit Mathur, Zuzana Berkova, and Haifeng Zhu
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Supplementary figure legends and materials
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- 2023
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4. Supplementary figure from HuR Suppresses Fas Expression and Correlates with Patient Outcome in Liver Cancer
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Felipe Samaniego, Asif Rashid, Boris Blechacz, Anita L. Sabichi, Lei Feng, Xue Ao, Rong-Hua Tao, Tamer Khashab, Lalit Sehgal, Rohit Mathur, Zuzana Berkova, and Haifeng Zhu
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Supplementary figure 1-2: Fig. S1. Fas mRNA in HCC cell lines and the effects of HuR knockdown on alternative Fas splicing. Fig S2. Effect of HuR knock-down on FasL-induced apoptosis in HepG2 cells.
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- 2023
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5. Data from Regulation of USP37 Expression by REST-Associated G9a-Dependent Histone Methylation
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Vidya Gopalakrishnan, Gianluca Sbardella, Pete H. Taylor, Sabrina Castellano, Ciro Milite, Rong-Hua Tao, Shavali Shaik, Chandra M. Das, Jyothishmathi Swaminathan, Rashieda J. Hatcher, and Tara H.W. Dobson
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The deubiquitylase (DUB) USP37 is a component of the ubiquitin system and controls cell proliferation by regulating the stability of the cyclin-dependent kinase inhibitor 1B, (CDKN1B/p27Kip1). The expression of USP37 is downregulated in human medulloblastoma tumor specimens. In the current study, we show that USP37 prevents medulloblastoma growth in mouse orthotopic models, suggesting that it has tumor-suppressive properties in this neural cancer. Here, we also report on the mechanism underlying USP37 loss in medulloblastoma. Previously, we observed that the expression of USP37 is transcriptionally repressed by the RE1 silencing transcription factor (REST), which requires chromatin remodeling factors for its activity. Genetic and pharmacologic approaches were employed to identify a specific role for G9a, a histone methyltransferase (HMT), in promoting methylation of histone H3 lysine-9 (H3K9) mono- and dimethylation, and surprisingly trimethylation, at the USP37 promoter to repress its gene expression. G9a inhibition also blocked the tumorigenic potential of medulloblastoma cells in vivo. Using isogenic low- and high-REST medulloblastoma cells, we further showed a REST-dependent elevation in G9a activity, which further increased mono- and trimethylation of histone H3K9, accompanied by downregulation of USP37 expression. Together, these findings reveal a role for REST-associated G9a and histone H3K9 methylation in the repression of USP37 expression in medulloblastoma.Implications: Reactivation of USP37 by G9a inhibition has the potential for therapeutic applications in REST-expressing medulloblastomas. Mol Cancer Res; 15(8); 1073–84. ©2017 AACR.
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- 2023
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6. Exercise Inhibits Doxorubicin-Induced Damage to Cardiac Vessels and Activation of Hippo/YAP-Mediated Apoptosis
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Rong-Hua Tao, Eugenie S. Kleinerman, Masato Kobayashi, and Yuanzheng Yang
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0301 basic medicine ,CD31 ,Cancer Research ,cardiotoxicity ,macromolecular substances ,doxorubicin ,Article ,pericytes ,Green fluorescent protein ,03 medical and health sciences ,0302 clinical medicine ,Vasculogenesis ,polycyclic compounds ,medicine ,Doxorubicin ,RC254-282 ,BM stem cells ,Cardiotoxicity ,exercise ,Chemistry ,organic chemicals ,apoptosis ,technology, industry, and agriculture ,Neoplasms. Tumors. Oncology. Including cancer and carcinogens ,endothelial cells ,Hippo-YAP signaling ,carbohydrates (lipids) ,030104 developmental biology ,medicine.anatomical_structure ,Oncology ,Apoptosis ,030220 oncology & carcinogenesis ,Cancer research ,Bone marrow ,Stem cell ,medicine.drug - Abstract
Dose-related cardiomyopathy is a major side effect following doxorubicin (Dox). To investigate whether exercise (Ex)-induced vasculogenesis plays a role in reducing Dox-induced cardiotoxicity, GFP+ bone marrow (BM) cells from GFP transgenic mice were transplanted into wild-type mice. Transplanted mice were treated with Dox, Ex, Dox+Ex, or control. We found Dox therapy resulted in decreased systolic and diastolic blood flow, decreased ejection fraction and fractional shortening, and decreased vascular endothelial cells and pericytes. These abnormalities were not seen in Dox+Ex hearts. Heart tissues from control-, Ex-, or Dox-treated mice showed a small number of GFP+ cells. By contrast, the Dox+Ex-treated hearts had a significant increase in GFP+ cells. Further analyses demonstrated these GFP+ BM cells had differentiated into vascular endothelial cells (GFP+CD31+) and pericytes (GFP+NG2+). Decreased cardiomyocytes were also seen in Dox-treated but not Dox+Ex-treated hearts. Ex induced an increase in GFP+c-Kit+ cells. However, these c-Kit+ BM stem cells had not differentiated into cardiomyocytes. Dox therapy induced phosphorylation of MST1/2, LATS1, and YAP, a decrease in total YAP, and cleavage of caspase-3 and PARP in the heart tissues. Dox+Ex prevented these effects. Our data demonstrated Dox-induced cardiotoxicity is mediated by vascular damage resulting in decreased cardiac blood flow and through activation of Hippo-YAP signaling resulting in cardiomyocyte apoptosis. Furthermore, Ex inhibited these effects by promoting migration of BM stem cells into the heart to repair the cardiac vessels damaged by Dox and through inhibiting Dox-induced Hippo-YAP signaling-mediated apoptosis. These data support the concept of using exercise as an intervention to decrease Dox-induced cardiotoxicity.
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- 2021
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7. Abstract 5422: Dissecting the mechanism of exercise-mediated protection of Dox-induced cardiotoxicity
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Eugenie S. Kleinerman, Fei Wang, Masato Kobayashi, Yuanzheng Yang, and Rong-Hua Tao
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Cardiac function curve ,CD31 ,Cancer Research ,Cardiotoxicity ,Chemistry ,Green fluorescent protein ,medicine.anatomical_structure ,Oncology ,Apoptosis ,polycyclic compounds ,medicine ,Cancer research ,Doxorubicin ,Bone marrow ,Stem cell ,medicine.drug - Abstract
Doxorubicin (Dox) is one of the most effective drugs for the treatment of childhood cancer patients. However, there is a correlation between Dox dose/intensity and cardiotoxicity. We developed a juvenile mouse cardiotoxicity model and showed that Dox treatment affected the cardiac vessel morphology characterized by punctate vessels with decreased pericytes and endothelial cells. Cardiac blood flow and function (as quantified by echocardiography [Echo]) were also compromised. These abnormalities were not seen in mice treated with exercise (Ex) during Dox. We also demonstrated increased c-kit+ cells in the hearts of mice treated with Dox + Ex. To determine the origin of these c-kit+ cells we performed a bone marrow (BM) transplant using GFP+ BM cells from C57BL/6Tg(UBC-GFP)30Scha/J mice into C57BL/6 WT mice treated with Busulfan and anti-CD4 and anti-DC8 Abs. BM engraftment was documented 4 wks post-transplant. Transplanted mice were treated with Dox twice/wk x 2 wks; Dox + Ex, or control (no treatment). Cardiac function was assessed after treatment by Echo and the heart tissues were evaluated by immunohistochemistry (IHC) for cells expressing GFP (denoting cells derived from the BM); GFP+/NG2+ or GFP+/CD31+ (BM cells differentiated into pericytes or endothelial cells respectively). Echo data confirmed decreased cardiac function in Dox but not Dox + Ex-treated mice. Few GFP+ cells were found in the hearts from control or Dox-treated mice. By contrast, mice treated with Dox + Ex showed a significant increase in GFP+ cells indicating that exercise had induced the migration of BM cells into the heart. Co-localization of GFP/NG2 and GFP/CD31 indicated that these BM stem cells had differentiated into both endothelial cells and pericytes, and that these cells were incorporated into the cardiac vessels. Few GFP+cTnI+ cells were observed in the heart tissues from mice treated with Ex + Dox, indicating that the BM stem cells had not differentiated into cardiomyoctes. These studies suggest that acute Dox-induced cardiac damage may be partially mediated by decreased blood flow secondary to damaged vessels with decreased vascular endothelial cells and pericytes. Ex may inhibit this effect by inducing the migration of BM stem cells to repair the damaged vessels. We also showed that Dox therapy induced phosphorylation of MST1/2, LATS1 and YAP, decreased total YAP, and induced cleavage of caspase-3 and PARP. These changes were not seen in Dox + Ex hearts suggesting that Ex protected cardiomyocytes from Dox-induced apoptosis by suppressing Hippo-YAP signaling. Taken together, our data demonstrated that the protective effect of Ex may be mediated by restoring cardiac blood flow which may in turn inhibit Dox-induced Hippo-YAP signaling-mediated cardiomyocyte apoptosis. These data support the concept that exercise interventions have the potential to decrease Dox-induced cardiac damage and morbidity in childhood cancer patients. Citation Format: Rong-Hua Tao, Masato Kobayashi, Fei Wang, Yuanzheng Yang, Eugenie S. Kleinerman. Dissecting the mechanism of exercise-mediated protection of Dox-induced cardiotoxicity [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5422.
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- 2020
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8. Transcriptional repressor REST drives lineage stage-specific chromatin compaction at
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Tara H W, Dobson, Rong-Hua, Tao, Jyothishmathi, Swaminathan, Shinji, Maegawa, Shavali, Shaik, Javiera, Bravo-Alegria, Ajay, Sharma, Bridget, Kennis, Yanwen, Yang, Keri, Callegari, Amanda R, Haltom, Pete, Taylor, Mari, Kogiso, Lin, Qi, Soumen, Khatua, Stewart, Goldman, Rishi R, Lulla, Jason, Fangusaro, Tobey J, MacDonald, Xiao-Nan, Li, Cynthia, Hawkins, Veena, Rajaram, and Vidya, Gopalakrishnan
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Adult ,Male ,Transplantation, Heterologous ,Mice, Transgenic ,Mice, SCID ,Article ,Mice, Inbred NOD ,Cell Line, Tumor ,Animals ,Humans ,Hedgehog Proteins ,Cerebellar Neoplasms ,Child ,Neoplasm Staging ,Mice, Knockout ,Infant ,Chromatin ,Gene Expression Regulation, Neoplastic ,Mice, Inbred C57BL ,Patched-1 Receptor ,Repressor Proteins ,Disease Models, Animal ,Female ,Proto-Oncogene Proteins c-akt ,Medulloblastoma ,Signal Transduction - Abstract
In medulloblastomas (MBs), the expression and activity of RE1-silencing transcription factor (REST) is increased in tumors driven by the sonic-hedgehog (SHH) pathway, specifically SHH-α (children 3–16 years) and SHH-β (infants) sub-groups. Unexpectedly, SHH-β tumors display more neuronal maturation compared to SHH-α tumors, yet both are correlated with poor overall patient survival. We studied the contribution of REST to tumorigenesis using a novel transgenic mouse model (REST(TG)). In this model, conditional NeuroD2-controlled REST transgene expression in lineage committed Patched 1 heterozygous (Ptch1(+/−)) cerebellar granule neuron progenitors (CGNPs) caused markedly accelerated tumorigenesis and penetrance and infiltrative disease. Mechanistic studies revealed a neuronal maturation context specific antagonistic interplay between the transcriptional repressor and activator, REST and Gli1, respectively. REST elevation and low expression of an inhibitor of Gli1, β-Arrestin1 (Arrb1), promoted Gli1 activity and Ptch1 expression in proliferating cells, in spite of increased histone H3K9 methylation at the Ptch1 promoter. However, in lineage committed REST(TG) CGNPs, Arrb1 increase and decreased Gli1 activity in conjunction with increased histone H3K9 methylationat the Ptch1 locus led to premature silencing of Ptch1 expression. In human tumors, PTCH1, GLI1 and ARRB1 expression were significantly decreased in SHH-β tumors compared to SHH-α tumors. These findings as well as the synergistic decrease of MB cell proliferation in culture upon pharmacological inhibition of G9a and histone deacetylase (HDAC) activities support a role for REST in MB progression in more lineage committed cells. Lineage committed REST(TG) CGNPs in comparison with proliferating progenitors, also exhibited decreased expression of the phosphatase tensin homolog (Pten), a negative regulator of Akt kinase. Consistent with this, human SHH-β tumors had significantly lower PTEN expression, although an unexpected decrease in its gene expression was seen in SHH-α tumors. Pharmacological blockade of AKT promoted apoptosis in REST-high cells in culture. Our findings linking REST to differentiation-specific chromatin remodeling, PTCH1 silencing, and AKT hyperactivation in MB tissues and models reveal potential subgroup specific therapeutic targets to explore for patients with SHH MB.
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- 2019
9. Transcriptional repressor REST drives lineage stage–specific chromatin compaction at Ptch1 and increases AKT activation in a mouse model of medulloblastoma
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Ajay Sharma, Shavali Shaik, Pete Taylor, Veena Rajaram, Cynthia Hawkins, Javiera Bravo-Alegria, Keri Callegari, Rishi Lulla, Yanwen Yang, Stewart Goldman, Tara Dobson, Lin Qi, Jason Fangusaro, Mari Kogiso, Vidya Gopalakrishnan, Xiao-Nan Li, Rong-Hua Tao, Soumen Khatua, Shinji Maegawa, Amanda R. Haltom, Bridget Kennis, Jyothishmathi Swaminathan, and Tobey J. MacDonald
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Regulation of gene expression ,0303 health sciences ,Cell Biology ,Biology ,Biochemistry ,Chromatin remodeling ,Chromatin ,Cell biology ,03 medical and health sciences ,0302 clinical medicine ,GLI1 ,030220 oncology & carcinogenesis ,biology.protein ,Gene silencing ,PTEN ,Sonic hedgehog ,Molecular Biology ,Protein kinase B ,030304 developmental biology - Abstract
In medulloblastomas (MBs), the expression and activity of RE1-silencing transcription factor (REST) is increased in tumors driven by the sonic hedgehog (SHH) pathway, specifically the SHH-α (children 3 to 16 years) and SHH-β (infants) subgroups. Neuronal maturation is greater in SHH-β than SHH-α tumors, but both correlate with poor overall patient survival. We studied the contribution of REST to MB using a transgenic mouse model ( RESTTG ) wherein conditional NeuroD2 -controlled REST transgene expression in lineage-committed Ptch1 +/− cerebellar granule neuron progenitors (CGNPs) accelerated tumorigenesis and increased penetrance and infiltrative disease. This model revealed a neuronal maturation context–specific antagonistic interplay between the transcriptional repressor REST and the activator GLI1 at Ptch1 . Expression of Arrb1 , which encodes β-arrestin1 (a GLI1 inhibitor), was substantially reduced in proliferating and, to a lesser extent, lineage-committed RESTTG cells compared with wild-type proliferating CGNPs. Lineage-committed REST TG cells also had decreased GLI1 activity and increased histone H3K9 methylation at the Ptch1 locus, which correlated with premature silencing of Ptch1 . These cells also had decreased expression of Pten , which encodes a negative regulator of the kinase AKT. Expression of PTCH1 and GLI1 were less, and ARRB1 was somewhat greater, in patient SHH-β than SHH-α MBs, whereas that of PTEN was similarly lower in both subtypes than in others. Inhibition of histone modifiers or AKT reduced proliferation and induced apoptosis, respectively, in cultured REST-high MB cells. Our findings linking REST to differentiation-specific chromatin remodeling, PTCH1 silencing, and AKT activation in MB tissues reveal potential subgroup-specific therapeutic targets for MB patients.
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- 2019
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10. Monitoring of intracerebellarly-administered natural killer cells with fluorine-19 MRI
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Nancy Gordon, Javiera Bravo-Alegria, David I. Sandberg, Vidya Gopalakrishnan, Rong-Hua Tao, Soumen Khatua, James A. Bankson, Laurence J.N. Cooper, Dean A. Lee, Wafik Zaky, Samuel A. Einstein, Shinji Maegawa, William Brugmann, Álvaro Macedo Laureano, Andrew Wahba, Keith A. Michel, Srinivas S. Somanchi, Dawid Schellingerhout, Bridget Kennis, Lucia Mariano da Rocha Silla, and Simin Kiany
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Cancer Research ,Fluorine Radioisotopes ,medicine.medical_treatment ,Apoptosis ,Mice, SCID ,03 medical and health sciences ,Mice ,0302 clinical medicine ,In vivo ,Mice, Inbred NOD ,medicine ,Tumor Cells, Cultured ,Bioluminescence imaging ,Animals ,Humans ,Cerebellar Neoplasms ,Cell Proliferation ,Medulloblastoma ,Chemistry ,Cell migration ,Immunotherapy ,medicine.disease ,Magnetic Resonance Imaging ,Xenograft Model Antitumor Assays ,In vitro ,Killer Cells, Natural ,Neurology ,Oncology ,Cell culture ,030220 oncology & carcinogenesis ,Cancer research ,Neurology (clinical) ,Drug Monitoring ,030217 neurology & neurosurgery - Abstract
Medulloblastoma (MB) is the most common malignant brain tumor in children. Recent studies have shown the ability of natural killer (NK) cells to lyse MB cell lines in vitro, but in vivo successes remain elusive and the efficacy and fate of NK cells in vivo remain unknown. To address these questions, we injected MB cells into the cerebellum of immunodeficient mice and examined tumor growth at various days after tumor establishment via bioluminescence imaging. NK cells were labeled with a fluorine-19 (19F) MRI probe and subsequently injected either intratumorally or contralaterally to the tumor in the cerebellum and effect on tumor growth was monitored. The 19F probe efficiently labeled the NK cells and exhibited little cytotoxicity. Fluorine-19 MRI confirmed the successful and accurate delivery of the labeled NK cells to the cerebellum of the mice. Administration of 19F–labeled NK cells suppressed MB growth, with the same efficacy as unlabeled cells. Immunohistochemistry confirmed the presence of NK cells within the tumor, which was associated with induction of apoptosis in tumor cells. NK cell migration to the tumor from a distal location as well as activation of apoptosis was also demonstrated by immunohstochemistry. Our results show that NK cells present a novel opportunity for new strategies in MB treatment. Further, 19F-labeled NK cells can suppress MB growth while enabling 19F MRI to provide imaging feedback that can facilitate study and optimization of therapeutic paradigms.
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- 2018
11. MBRS-62. REPRESSIVE CHROMATIN REMODELERS IN SHH-DRIVEN MEDULLOBLASTOMA
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Vidya Gopalakrishnan, Xiao-Nan Li, Bridget Kennis, Rishi Lulla, Lin Qi, Jason Fangusaro, Shavali Shaik, Stewart Goldman, Pete Taylor, Veena Rajaram, Soumen Khatua, Cynthia Hawkins, Tobey J. MacDonald, Rong-Hua Tao, Tara Dobson, Shinji Maegawa, and Jyothismathi Swaminathan
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Medulloblastoma ,Cancer Research ,Biology ,medicine.disease ,Chromatin ,Cell biology ,Abstracts ,Oncology ,Gene expression ,Neuron differentiation ,medicine ,Neurology (clinical) ,Signal transduction ,Candidate Disease Gene ,Transcription factor ,Neural development - Abstract
Medulloblastoma (MB) is a malignant pediatric brain tumor characterized by poor neuronal differentiation. The RE1 Silencing Transcription Factor (REST), which negatively regulates neurogenesis by promoting repressive chromatin remodeling via epigenetic modifications, is overexpressed in human MBs. Analyses of a publically available gene expression array data set revealed association of elevated REST expression in patients with Sonic Hedgehog (SHH) medulloblastoma with poor prognosis. Upon closer evaluation, we identified a subset of highly undifferentiated SHHα tumors with elevated REST expression and activity as determined by expression of REST target genes. These patients had an overall survival rate of 50% at 5 years. To understand the underlying mechanisms, we generated a novel mouse model expressing human REST transgene in a conditional manner in granule neural progenitors (GNPs), the cells of origin of SHH driven MB. Mice with constitutive activation of Shh signaling and REST elevation (Ptch(+/-)/REST(TG)) in their GNPs developed tumors with a significant decrease in latency and increase in penetrance. We observed that 100% of Ptch(+/-)/REST(TG) animals developed heterogeneously proliferative and undifferentiated tumors compared to tumor-bearing Ptch(+/-) animals. Mechanistic studies revealed deregulation of the Shh signaling pathway by REST-dependent G9a activity, accompanied by loss of Ptch1 heterozygosity in Ptch(+/-)/REST(TG) tumors. Thus, our studies suggest that REST contributes to mis-regulation of SHH activity and drives an aggressive disease course by deregulating proliferation and differentiation. Ours is also the first study to implicate G9a, a repressive chromatin remodeler, in medulloblastoma pathogenesis and as a potential therapeutic target.
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- 2018
12. HuR Suppresses Fas Expression and Correlates with Patient Outcome in Liver Cancer
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Asif Rashid, Lalit Sehgal, Xue Ao, Anita L. Sabichi, Zuzana Berkova, Rohit Mathur, Boris Blechacz, Felipe Samaniego, Lei Feng, Tamer Khashab, Haifeng Zhu, and Rong-Hua Tao
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Male ,Cancer Research ,Carcinoma, Hepatocellular ,Fas Ligand Protein ,Cell Survival ,Cell ,Biology ,Transfection ,Fas ligand ,ELAV-Like Protein 1 ,Mice ,Downregulation and upregulation ,Cell Line, Tumor ,Translational regulation ,medicine ,Animals ,Humans ,Gene silencing ,RNA, Messenger ,fas Receptor ,Molecular Biology ,Cell Death ,Liver Neoplasms ,Fas receptor ,Molecular biology ,digestive system diseases ,Mice, Inbred C57BL ,medicine.anatomical_structure ,Oncology ,Apoptosis ,Gene Knockdown Techniques ,Protein Biosynthesis ,Cancer research ,Female - Abstract
Hepatocellular carcinomas (HCC) show resistance to chemotherapy and have blunt response to apoptotic stimuli. HCC cell lines express low levels of the Fas death receptor and are resistant to FasL stimulation, whereas immortalized hepatocytes are sensitive. The variable Fas transcript levels and consistently low Fas protein in HCC cells suggest posttranscriptional regulation of Fas expression. The 3′-untranslated region (UTR) of Fas mRNA was found to interact with the ribonucleoprotein Human Antigen R (HuR) to block mRNA translation. Silencing of HuR in HCC cells increased the levels of cell surface Fas and sensitized HCC cells to FasL. Two AU-rich domains within the 3′-UTR of Fas mRNA were identified as putative HuR-binding sites and were found to mediate the translational regulation in reporter assay. Hydrodynamic transfection of HuR plasmid into mice induced downregulation of Fas expression in livers and established functional resistance to the killing effects of Fas agonist. Human HCC tumor tissues showed significantly higher overall and cytoplasmic HuR staining compared with normal liver tissues, and the high HuR staining score correlated with worse survival of patients with early-stage HCC. Combined, the protumorigenic ribonucleoprotein HuR blocks the translation of Fas mRNA and effectively prevents Fas-mediated apoptosis in HCC, suggesting that targeting HuR would sensitize cells to apoptotic stimuli and reverse tumorigenic properties. Implications: Demonstrating how death receptor signaling pathways are altered during progression of HCC will enable the development of better methods to restore this potent apoptosis mechanism. Mol Cancer Res; 13(5); 809–18. ©2015 AACR.
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- 2015
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13. Medulloblastoma development: tumor biology informs treatment decisions
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Vidya Gopalakrishnan, William Brugmann, Soumen Khatua, Tara Dobson, and Rong-Hua Tao
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Oncology ,Medulloblastoma ,Clinical Trials as Topic ,medicine.medical_specialty ,Brain Neoplasms ,Tumor biology ,business.industry ,Clinical Decision-Making ,Personalized treatment ,General Medicine ,medicine.disease ,Article ,Treatment modality ,Internal medicine ,Immunology ,medicine ,Pediatric Brain Tumor ,Animals ,Humans ,Treatment decision making ,business ,Craniospinal ,Neurocognitive - Abstract
SUMMARY Medulloblastoma is the most common malignant pediatric brain tumor. Current treatments including surgery, craniospinal radiation and high-dose chemotherapy have led to improvement in survival. However, the risk for recurrence as well as significant long-term neurocognitive and endocrine sequelae associated with current treatment modalities underscore the urgent need for novel tumor-specific, normal brain-sparing therapies. It has also provided the impetus for research focused on providing a better understanding of medulloblastoma biology. The expectation is that such studies will lead to the identification of new therapeutic targets and eventually to an increase in personalized treatment approaches.
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- 2015
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14. Regulation of USP37 Expression by REST-associated G9a-dependent Histone Methylation
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Rashieda Hatcher, Rong-Hua Tao, Shavali Shaik, Pete Taylor, Jyothishmathi Swaminathan, Sabrina Castellano, Tara Dobson, Vidya Gopalakrishnan, Ciro Milite, Gianluca Sbardella, and Chandra M. Das
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0301 basic medicine ,Cancer Research ,Methyltransferase ,G9a ,Carcinogenesis ,RE1-silencing transcription factor ,Biology ,Methylation ,Chromatin remodeling ,Article ,Histones ,03 medical and health sciences ,Histone H3 ,Mice ,Histocompatibility Antigens ,Histone methylation ,Histone H2A ,Endopeptidases ,Animals ,Humans ,Molecular Biology ,Cancer ,Cell Proliferation ,Deubiquitinase USP37 ,Ubiquitin ,Protein ,Cyclin-dependent kinase ,Histone-Lysine N-Methyltransferase ,Methyltransferases ,Fission yeast ,Xenograft Model Antitumor Assays ,Gene Expression Regulation, Neoplastic ,Repressor Proteins ,H3 ,030104 developmental biology ,Histone ,c-Myc ,Oncology ,Histone methyltransferase ,Cancer research ,biology.protein ,Medulloblastoma - Abstract
The deubiquitylase (DUB) USP37 is a component of the ubiquitin system and controls cell proliferation by regulating the stability of the cyclin-dependent kinase inhibitor 1B, (CDKN1B/p27Kip1). The expression of USP37 is downregulated in human medulloblastoma tumor specimens. In the current study, we show that USP37 prevents medulloblastoma growth in mouse orthotopic models, suggesting that it has tumor-suppressive properties in this neural cancer. Here, we also report on the mechanism underlying USP37 loss in medulloblastoma. Previously, we observed that the expression of USP37 is transcriptionally repressed by the RE1 silencing transcription factor (REST), which requires chromatin remodeling factors for its activity. Genetic and pharmacologic approaches were employed to identify a specific role for G9a, a histone methyltransferase (HMT), in promoting methylation of histone H3 lysine-9 (H3K9) mono- and dimethylation, and surprisingly trimethylation, at the USP37 promoter to repress its gene expression. G9a inhibition also blocked the tumorigenic potential of medulloblastoma cells in vivo. Using isogenic low- and high-REST medulloblastoma cells, we further showed a REST-dependent elevation in G9a activity, which further increased mono- and trimethylation of histone H3K9, accompanied by downregulation of USP37 expression. Together, these findings reveal a role for REST-associated G9a and histone H3K9 methylation in the repression of USP37 expression in medulloblastoma. Implications: Reactivation of USP37 by G9a inhibition has the potential for therapeutic applications in REST-expressing medulloblastomas. Mol Cancer Res; 15(8); 1073–84. ©2017 AACR.
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- 2017
15. Authentication Algorithm Based on Hash-Tree for Web Single Sign-On
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Dong Ren, Qiang Wei, Rong Hua Tao, and Ze Hui Wu
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Challenge-Handshake Authentication Protocol ,Authentication ,Computer science ,Email authentication ,General Medicine ,Multi-factor authentication ,Login ,Computer security ,computer.software_genre ,Hash-based message authentication code ,ComputingMilieux_MANAGEMENTOFCOMPUTINGANDINFORMATIONSYSTEMS ,Generic Bootstrapping Architecture ,Authentication protocol ,Lightweight Extensible Authentication Protocol ,Single sign-on ,Challenge–response authentication ,Algorithm ,computer ,Data Authentication Algorithm - Abstract
During the authentication process of web-based single sign-on system, it is insecure that all authentication messages are forwarded by the browser, and its integrity protection is not comprehensive. This vulnerability can be exploited by attackers to bypass the authentication systems, login any account. In this work we analyze the vulnerability threat model and its root causes in detail, and propose an authentication algorithm based on Hash-tree. This algorithm can not only improve the security of the system, but the processing efficiency of the system is also acceptable according to the simulation results.
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- 2014
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16. Implementation of Bar Chart Plotting Based on IDL Object Graphics
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Xiao Lu Chen, Rong Hua Tao, and Biao Chen
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Engineering drawing ,Bar chart ,law ,Computer science ,Computer graphics (images) ,Graph (abstract data type) ,Functional requirement ,General Medicine ,Graphics ,law.invention ,Visualization - Abstract
Bar chart is the most common graph when we need to process some data counting, analysis and comparing. But there is no inner bar chart object in IDL object graphics. According to the outlook demands and the functional requirements of bar char object, a visual expression method of bar char object was implemented based on the inner basic graphics objects in IDL. The implementation method was presented in detail. The results show it can meet the requirements of bar char displaying of common information, and it is effective and extendable.
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- 2012
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17. PMLRARα binds to Fas and suppresses Fas-mediated apoptosis through recruiting c-FLIP in vivo
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David H. Hawke, Rong-Hua Tao, Jillian F. Wise, Urszula Daniluk, Xue Ao, Zuzana Berkova, Judith E. Karp, Hui Kuan Lin, Jeffrey J. Molldrem, Felipe Samaniego, and Abdol Hossein Rezaeian
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Acute promyelocytic leukemia ,Oncogene Proteins, Fusion ,Immunology ,CASP8 and FADD-Like Apoptosis Regulating Protein ,Down-Regulation ,Apoptosis ,HL-60 Cells ,Mice, Transgenic ,Biology ,Models, Biological ,Biochemistry ,Fas ligand ,Mice ,Promyelocytic leukemia protein ,medicine ,Animals ,Humans ,fas Receptor ,Cells, Cultured ,Death domain ,Myeloid Neoplasia ,U937 Cells ,Cell Biology ,Hematology ,Fas receptor ,medicine.disease ,Cell biology ,Mice, Inbred C57BL ,Cancer cell ,biology.protein ,Female ,Signal transduction ,Protein Binding - Abstract
Defective Fas signaling leads to resistance to various anticancer therapies. Presence of potential inhibitors of Fas which could block Fas signaling can explain cancer cells resistance to apoptosis. We identified promyelocytic leukemia protein (PML) as a Fas-interacting protein using mass spectrometry analysis. The function of PML is blocked by its dominant-negative form PML–retinoic acid receptor α (PMLRARα). We found PMLRARα interaction with Fas in acute promyelocytic leukemia (APL)–derived cells and APL primary cells, and PML-Fas complexes in normal tissues. Binding of PMLRARα to Fas was mapped to the B-box domain of PML moiety and death domain of Fas. PMLRARα blockage of Fas apoptosis was demonstrated in U937/PR9 cells, human APL cells and transgenic mouse APL cells, in which PMLRARα recruited c-FLIPL/S and excluded procaspase 8 from Fas death signaling complex. PMLRARα expression in mice protected the mice against a lethal dose of agonistic anti-Fas antibody (P < .001) and the protected tissues contained Fas-PMLRARα-cFLIP complexes. Taken together, PMLRARα binds to Fas and blocks Fas-mediated apoptosis in APL by forming an apoptotic inhibitory complex with c-FLIP. The presence of PML-Fas complexes across different tissues implicates that PML functions in apoptosis regulation and tumor suppression are mediated by direct interaction with Fas.
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- 2011
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18. Milatuzumab – a promising new immunotherapeutic agent
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Rong-Hua Tao, Felipe Samaniego, and Zuzana Berkova
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CD74 ,medicine.medical_treatment ,Chronic lymphocytic leukemia ,Drug Evaluation, Preclinical ,Antineoplastic Agents ,Biology ,Pharmacology ,Antibodies, Monoclonal, Humanized ,chemistry.chemical_compound ,Neoplasms ,medicine ,Animals ,Humans ,Pharmacology (medical) ,Multiple myeloma ,Clinical Trials as Topic ,Histocompatibility Antigens Class II ,Antibodies, Monoclonal ,General Medicine ,Immunotherapy ,medicine.disease ,Lymphoma ,Milatuzumab ,Antigens, Differentiation, B-Lymphocyte ,chemistry ,Cancer cell ,Monoclonal ,Cancer research - Abstract
Milatuzumab is a new immunotherapeutic agent targeting CD74, a membrane protein preferentially expressed in hematopoietic cancers and some solid tumors. Broad expression and fast internalization makes CD74 an ideal target for cancer therapy. We reviewed published articles about CD74 and milatuzumab. We present a comprehensive review of CD74 functions and provide explanation of milatuzumab antitumor effects. This review describes CD74 protein biology with the emphasis on the role of CD74 in tumor survival and its new role in regulation of the Fas death receptor. The development of CD74 targeting therapies to induce tumor regression and cancer cell apoptosis is described and results of clinical trials are discussed. Milatuzumab shows selective binding and rapid internalization into CD74-positive cancer cells. Milatuzumab with and without conjugated toxins synergizes with other chemotherapeutic agents and elicits significant antitumor effects in mice. In a Phase I trial, milatuzumab showed no severe adverse effects in patients with relapsed/refractory multiple myeloma and it stabilized the disease in some patients for up to 12 weeks. Ongoing trials testing different treatment schedules of milatuzumab in chronic lymphocytic leukemia, non-Hodgkin's lymphoma and multiple myeloma indicate that milatuzumab shows no severe adverse effects in humans.
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- 2009
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19. Testicular zinc finger protein recruits histone deacetylase 2 and suppresses the transactivation function and intranuclear foci formation of agonist-bound androgen receptor competitively with TIF2
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Atsuto Inoue, Ryoichi Takayanagi, Hisaya Kawate, Rong-Hua Tao, Yin Wu, Masamichi Ishizuka, Keizo Ohnaka, and Hiromi Hagiwara
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Male ,Transcriptional Activation ,Agonist ,medicine.drug_class ,Histone Deacetylase 2 ,Biology ,Biochemistry ,Histone Deacetylases ,Cell Line ,Green fluorescent protein ,Mice ,Nuclear Receptor Coactivator 2 ,Transactivation ,Endocrinology ,Chlorocebus aethiops ,Coactivator ,medicine ,Animals ,Humans ,Cloning, Molecular ,Molecular Biology ,Zinc finger ,Histone deacetylase 2 ,5-alpha-Dihydroprogesterone ,Cell biology ,Repressor Proteins ,Androgen receptor ,Receptors, Androgen ,Mutation ,Androgens ,Cancer research ,Corepressor ,Protein Binding - Abstract
We previously reported that testicular zinc finger protein (TZF) is a corepressor for androgen receptor (AR). The present study demonstrated that a central portion (amino acids 512–663) of TZF, TZF(512–663), is responsible for both binding to AR and repressing the transactivation. TZF recruited endogenous histone deacetylase 2 (HDAC2) and formed a complex with agonist-bound AR. Imaging analyses showed that TZF and TZF(512–663) were recruited by AR and simultaneously impaired distinct AR foci formation. Quantification of the foci number using a three-dimensional imaging method revealed that the number of intranuclear AR foci was related to its transactivation activity. Moreover, increased levels of TZF dissociated a coactivator, TIF2, from the AR foci and vice versa. These results indicate that the ligand-dependent transactivation function of AR is quantitatively related to its intranuclear foci formation, and suggest that corepressors, such as TZF, act on these intranuclear events competitively with coactivators.
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- 2006
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20. Impaired Nuclear Translocation, Nuclear Matrix Targeting, and Intranuclear Mobility of Mutant Androgen Receptors Carrying Amino Acid Substitutions in the Deoxyribonucleic Acid-Binding Domain Derived from Androgen Insensitivity Syndrome Patients
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Ryoichi Takayanagi, Rong-Hua Tao, Taijiro Okabe, Keizo Ohnaka, Hisaya Kawate, Yin Wu, Kei Ichiro Nakamura, Hajime Nawata, and Toshihiko Yanase
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Male ,medicine.medical_specialty ,medicine.drug_class ,Endocrinology, Diabetes and Metabolism ,Molecular Sequence Data ,Clinical Biochemistry ,Active Transport, Cell Nucleus ,Biology ,Biochemistry ,Transactivation ,Endocrinology ,Internal medicine ,Chlorocebus aethiops ,medicine ,Animals ,Humans ,Nuclear Matrix ,Amino Acid Sequence ,Receptor ,Zinc finger ,Binding Sites ,Biochemistry (medical) ,DNA ,Androgen-Insensitivity Syndrome ,Nuclear matrix ,medicine.disease ,Androgen ,Androgen receptor ,Amino Acid Substitution ,Receptors, Androgen ,COS Cells ,Mutation ,Androgen insensitivity syndrome ,Binding domain - Abstract
Context: Recent imaging studies revealed that androgen receptor (AR) is ligand-dependently translocated from the cytoplasm into the nucleus and forms intranuclear fine foci. In this study, we examined whether intracellular dynamics of mutant ARs detected in two androgen insensitivity syndrome (AIS) patients was impaired.Objective: ARs with mutations in the DNA-binding domain were functionally characterized and compared with the wild-type AR.Patients: In a complete AIS patient (subject 1), cysteine residue 579 in the first zinc finger motif of AR was substituted for phenylalanine (AR-C579F). Another mutation (AR-F582Y) was found in a partial AIS patient (subject 2).Results: AR-F582Y retained less than 10% of the transactivation activity of the wild-type AR, whereas no ligand-dependent transactivation was detected for AR-C579F. Image analyses of the receptors fused to green fluorescent protein showed that the wild-type AR was ligand-dependently translocated into the nucleus in which it formed fine subnuclear foci. Surprisingly, after the addition of dihydrotestosterone, the two mutant ARs initially formed large cytoplasmic dots, many of which were found to be close to mitochondria by electron microscopy. Subsequently, a part of the ligand-bound mutant ARs gradually entered the nucleus to form a smaller number of larger dots, compared with the wild-type AR. Fluorescence recovery after photobleaching analysis revealed that the intranuclear mobility of the mutant ARs decreased, compared with that of the wild-type AR.Conclusions: These results suggest that the abnormal translocation, localization, and mobility of the mutant ARs may be the cause of AIS in these subjects.
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- 2005
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21. A zinc finger protein TZF is a novel corepressor of androgen receptor
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Hisaya Kawate, Masamichi Ishizuka, Ryoichi Takayanagi, Hirotaka Ohshima, Hiromi Hagiwara, and Rong-Hua Tao
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Transcriptional Activation ,Zinc finger ,RNA Splicing ,Biophysics ,Repressor ,Zinc Fingers ,Cell Biology ,Biology ,Biochemistry ,Molecular biology ,Fusion protein ,Repressor Proteins ,Androgen receptor ,Nuclear receptor ,Receptors, Androgen ,COS Cells ,LNCaP ,Androgen Receptor Antagonists ,Animals ,Nuclear protein ,Molecular Biology ,Corepressor ,Protein Binding - Abstract
Steroid hormones control the transcriptional activity of target genes mediated by intracellular nuclear receptors, and these transcriptional activities are modulated by the combination with coactivators and corepressors. We found in this study that testicular zinc finger protein (TZF) that was a nuclear protein with a zinc finger motif of the Cys2-His2 type was a novel corepressor of androgen receptor (AR). Fusion protein with green fluorescence protein GFP formed the specific foci in nuclei and TZF-dependent foci were located close to the splicing factor compartment. In addition, TZF was recruited into AR subnuclear foci after the treatment of dihydrotestosterone. Furthermore, we revealed that TZF bound to the activation function-1 (AF-1) domain (N-terminal transactivating domain) of AR protein. Transient over-expression of TZF in COS-7 cells or LNCaP human prostatic cancer cell resulted in decreased AR activity in a ligand-dependent fashion. Moreover, a transcriptional corepressor N-CoR additively decreased the transcriptional activity of AR with TZF. These findings suggest that TZF might be a novel corepressor of AR.
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- 2005
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22. Nucleolin inhibits Fas ligand binding and suppresses Fas-mediated apoptosis in vivo via a surface nucleolin-Fas complex
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Xue Ao, Felipe Samaniego, Anita L. Sabichi, Rohit Mathur, Frank K. Braun, Rong-Hua Tao, Hoyoung Maeng, Jillian F. Wise, Haifeng Zhu, and Zuzana Berkova
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Fas Ligand Protein ,Lymphoma, B-Cell ,Immunology ,Cell ,Blotting, Western ,Molecular Sequence Data ,Apoptosis ,Biology ,Real-Time Polymerase Chain Reaction ,Biochemistry ,Fas ligand ,Mice ,medicine ,Tumor Cells, Cultured ,Animals ,Humans ,Immunoprecipitation ,Amino Acid Sequence ,RNA, Messenger ,Cell Proliferation ,B-Lymphocytes ,Lymphoid Neoplasia ,Cell growth ,Reverse Transcriptase Polymerase Chain Reaction ,Cell Membrane ,RNA-Binding Proteins ,Cell Biology ,Hematology ,Transfection ,Fas receptor ,Flow Cytometry ,Phosphoproteins ,Mice, Inbred C57BL ,medicine.anatomical_structure ,Cancer research ,Signal transduction ,Nucleolin ,Protein Binding ,Signal Transduction - Abstract
Resistance to Fas-mediated apoptosis is associated with poor cancer outcomes and chemoresistance. To elucidate potential mechanisms of defective Fas signaling, we screened primary lymphoma cell extracts for Fas-associated proteins that would have the potential to regulate Fas signaling. An activation-resistant Fas complex selectively included nucleolin. We confirmed the presence of nucleolin-Fas complexes in B-cell lymphoma cells and primary tissues, and the absence of such complexes in B-lymphocytes from healthy donors. RNA-binding domain 4 and the glycine/arginine-rich domain of nucleolin were essential for its association with Fas. Nucleolin colocalized with Fas on the surface of B-cell lymphoma cells. Nucleolin knockdown sensitized BJAB cells to Fas ligand (FasL)-induced and Fas agonistic antibody-induced apoptosis through enhanced binding, suggesting that nucleolin blocks the FasL–Fas interaction. Mice transfected with nucleolin were protected from the lethal effects of agonistic anti-mouse Fas antibody (Jo2) and had lower rates of hepatocyte apoptosis, compared with vector and a non-Fas-binding mutant of nucleolin. Our results show that cell surface nucleolin binds Fas, inhibits ligand binding, and thus prevents induction of Fas-mediated apoptosis in B-cell lymphomas and may serve as a new therapeutic target.
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- 2013
23. MB-107REST ELEVATION UNRESTRAINS SHH SIGNALING IN MEDULLOBLASTOMA
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Stewart Goldman, Ellen Busschers, Bridget Kennis, Vidya Gopalakrishnan, Tobey J. MacDonald, Tara Dobson, William Brugmann, Jason Fangusaro, Rishi Lulla, Shavali Shaik, Pete Taylor, Rashieda Hatcher, Cynthia Hawkins, Soumen Khatua, Veena Rajaram, Wafik Zaky, and Rong-Hua Tao
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Medulloblastoma ,Cancer Research ,business.industry ,Elevation ,Biology ,medicine.disease ,Abstracts ,Text mining ,Oncology ,Shh signaling ,medicine ,Cancer research ,Neurology (clinical) ,Signal transduction ,business - Published
- 2016
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24. All EGF(ErbB) receptors have preformed homo- and heterodimeric structures in living cells
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Rong-Hua Tao and Ichi N. Maruyama
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Cell Survival ,Intracellular Space ,CHO Cells ,Endoplasmic Reticulum ,Ligands ,Models, Biological ,Receptor tyrosine kinase ,Fluorescence ,ErbB Receptors ,Mice ,Cricetulus ,ErbB ,Cell Line, Tumor ,Cricetinae ,Animals ,Humans ,ERBB3 ,Epidermal growth factor receptor ,Receptor ,Cell Nucleus ,biology ,Cell Biology ,Ligand (biochemistry) ,Cell biology ,Protein Structure, Tertiary ,Protein Transport ,Biochemistry ,biology.protein ,NIH 3T3 Cells ,Signal transduction ,Protein Multimerization ,Subcellular Fractions - Abstract
The epidermal growth factor receptor (EGFR) family of receptor tyrosine kinases, also known as ErbB or HER, plays crucial roles in the development of multicellular organisms. Mutations and over-expression of the ErbB receptors have been implicated in a variety of human cancers. It is widely thought that the ErbB receptors are located in the plasma membrane, and that ligand binding to the monomeric form of the receptors induces its dimeric form for activation. However, it still remains controversial whether prior to ligand binding the receptors exist as monomers or dimers on the cell surface. Using bimolecular fluorescence complementation (BiFC) assays in the present study, we demonstrate that in the absence of bound ligand, all the ErbB family members have preformed, yet inactive, homo- and heterodimers on the cell surface, except for ErbB3 homodimers and heterodimers with cleavable ErbB4, which exist primarily in the nucleus. BiFC assays of the dimerization have also suggested that the ligand-independent dimerization of the ErbB receptors occurs in the endoplasmic reticulum (ER) before newly synthesized receptor molecules reach the cell surface. Based on BiFC and mammalian two-hybrid assays, it is apparent that the intracellular domains of the receptors are responsible for the spontaneous dimer formation. These provide new insights into an understanding of transmembrane signal transduction mediated by the ErbB family members, and are relevant to the development of anti-cancer drugs.
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- 2008
25. Ligand‐induced activation of preformed inactive EGF/ErbB receptor homo‐ and heterodimers: a model for EGF/ErbB receptors
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Rong-Hua Tao and Ichi N. Maruyama
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ErbB Receptors ,ErbB ,Chemistry ,Genetics ,Cancer research ,Receptor ,Ligand (biochemistry) ,Molecular Biology ,Biochemistry ,Biotechnology - Published
- 2008
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26. Cell‐surface and nuclear localization of preformed ErbB receptor homo‐ and heterodimers in living cells
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Rong-Hua Tao and Ichi N. Maruyama
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medicine.anatomical_structure ,Biochemistry ,Chemistry ,ErbB ,Cell ,Genetics ,medicine ,Receptor ,Molecular Biology ,Nuclear localization sequence ,Biotechnology ,Cell biology - Published
- 2007
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27. MB-29 * DISSECTING THE ROLE OF EPIGENETIC MODULATORS IN SHH-DRIVEN MEDULLOBLASTOMA
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Shavali Shaik, Pete Taylor, Rong-Hua Tao, Jason Fangusaro, Vidya Gopalakrishnan, Soumen Khatua, Rishi Lulla, Tobey J. MacDonald, Veena Rajaram, Ellen Busschers, Rashieda Hatcher, Stewart Goldman, and Tara Dobson
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Medulloblastoma ,Cancer Research ,medicine.medical_specialty ,endocrine system diseases ,biology ,Transgene ,Neurogenesis ,Context (language use) ,Vimentin ,Nestin ,medicine.disease ,Neural stem cell ,Endocrinology ,Oncology ,Internal medicine ,medicine ,Cancer research ,biology.protein ,Neurology (clinical) ,Sonic hedgehog ,Abstracts from the 3rd Biennial Conference on Pediatric Neuro-Oncology Basic and Translational Research - Abstract
The Repressor Element-1 Silencing Transcription Factor (REST) negatively regulates neurogenesis. It epigenetically silences the expression of neuronal differentiation genes through modulation of histone acetylation and histone methylation. REST levels are elevated in over 80% of human medulloblastomas, and spans all four molecular sub-groups. Interestingly, a subset of patients with sonic hedgehog (Shh)-driven medulloblastomas with high REST expression (+3/ + 4 on a scale of 0-4) had a significantly elevated incidence of metastatic disease compared to patients with low-REST or no-REST expressing Shh-driven tumors. To understand if REST is a driver of metastatic disease, we generated a novel REST knock-in (RESTKI) mouse model in which transgene expression is conditionally elevated in cerebellar granule neuron progenitors (GNPs), the cells of origin of Shh-driven medulloblastomas. RESTKI mice were then crossed with mice exhibiting constitutive activation of Shh signaling (Ptc +/−), and REST transgene expression was induced in the progeny (RESTKI/Ptc +/−) on postnatal day 2. While RESTKI mice exhibited pre-neoplastic lesions, both Ptc +/− and RESTKI/Ptc +/− mice formed cerebellar tumors. However, a significant increase in tumor penetrance (100%) was seen in RESTKI/Ptc +/− mice compared to 20% in Ptc +/− mice. A substantial decrease in tumor latency (10-40 days) was also observed in RESTKI/Ptc +/− in contrast to 3-7 months in Ptc +/− mice. Histopathological analyses of mice brains revealed leptomeningeal and infiltrative medulloblastoma with significant changes in the tumor microenvironment in RESTKI/Ptc +/− mice. In contrast, cerebella of Ptc +/− mice exhibited localized disease. Importantly, RESTKI/ Ptc +/− tumors expressed very high levels of neural stem cell (NSC) markers such as Nestin, GFAP and Vimentin when compared with Ptc +/− tumors, suggesting that REST elevation in GNPs may have resulted in their de-differentiation into a more primitive state, and driven medulloblastoma genesis in the context of Shh hyperactivation. Our studies implicate REST in the modulation of the oncogenic potential of Shh signaling and medulloblastoma development.
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- 2015
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28. IM-07 * NK CELL IMMUNOTHERAPY FOR PEDIATRIC BRAIN TUMORS: OVERCOMING RESISTANCE TO EXPAND THERAPEUTIC SUCCESS
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Laurence J.N. Cooper, Helen Huls, William Brugmann, King Ching Ho, Keith A. Michel, Lucia Mariano da Rocha Silla, Álvaro Macedo Laureano, Rong-Hua Tao, Annie Huang, David I. Sandberg, Srinivas S. Somanchi, Harjeet Singh, Vidya Gopalakrishnan, Dean A. Lee, Cecele J. Denman, James A. Bankson, and Bridget Kennis
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Medulloblastoma ,Cancer Research ,Chemotherapy ,business.industry ,medicine.medical_treatment ,Cell ,Brain tumor ,Phases of clinical research ,Immunotherapy ,medicine.disease ,medicine.anatomical_structure ,Oncology ,Antigen ,Cell culture ,Immunology ,Cancer research ,Medicine ,Neurology (clinical) ,business ,Abstracts from the 3rd Biennial Conference on Pediatric Neuro-Oncology Basic and Translational Research - Abstract
Pediatric brain tumors including medulloblastomas and ATRTs are associated with significant mortality and morbidity. Much of this morbidity is due to conventional treatments: radiation and chemotherapy. Therefore, there is an urgent need to develop new therapeutic options to combat these devastating diseases. Immunotherapy has gained traction as a potential alternative to current treatments. Because many immunotherapies rely on the presence of tumor-associated antigens (TAAs) and pediatric brain tumors have poorly defined antigen profiles, we pursued the development of natural killer (NK) cells, which don't require TAAs for their activity, to treat these malignancies. Our work shows that most medulloblastoma and ATRT cell lines are sensitive to NK cell lysis in vitro. Furthermore, both intratumoral NK cell injections as well as infusion at a distant site limit the medulloblastoma growth in mouse orthotopic xenograft models, indicating NK cell trafficking through the brain. These results have provided the foundation for a FDA-approved Phase I clinical trial to infuse NK cells directly into the fourth ventricle of patients who have undergone re-resection of infratentorial tumors. The trial is scheduled to enroll patients in February 2015. Interestingly, our pre-clinical data has also identified overexpression of a novel tumor-secreted immunosuppressive molecule, which was originally shown to confer cardio-protection following myocardial ischemia, in human ATRT samples, and its involvement in promoting resistance to NK cell-mediated lysis. In conclusion, our findings have paved the way for a first in pediatrics brain tumor immunotherapy trial that merges two cutting edge technologies: immunotherapy and loco-regional therapeutics delivery. Our study also offers a novel biomarker for predicting patient response to NK therapy.
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- 2015
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29. Opposite effects of alternative TZF spliced variants on androgen receptor
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Rong-Hua Tao, Masamichi Ishizuka, Keizo Ohnaka, Ryoichi Takayanagi, Hisaya Kawate, and Hiromi Hagiwara
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Gene isoform ,Transcriptional Activation ,Amino Acid Motifs ,Immunoblotting ,Biophysics ,Biology ,Transfection ,Biochemistry ,Models, Biological ,Green fluorescent protein ,Transactivation ,Mice ,Transcription (biology) ,Genes, Reporter ,Cell Line, Tumor ,Coactivator ,Chlorocebus aethiops ,Animals ,Humans ,Immunoprecipitation ,Protein Isoforms ,Molecular Biology ,Microscopy, Confocal ,Cell Biology ,Molecular biology ,Protein Structure, Tertiary ,Androgen receptor ,Repressor Proteins ,Alternative Splicing ,Receptors, Androgen ,COS Cells ,NIH 3T3 Cells ,Corepressor ,Dimerization ,Gene Deletion ,Plasmids - Abstract
We previously demonstrated that testicular zinc-finger protein (TZF) was a corepressor of the androgen receptor (AR). In the present study, we further showed that TZF-L, an alternative spliced variant of TZF, enhanced transactivation function of AR. Deletion analysis of TZF-L revealed that its N-terminus, which almost corresponded to that of TZF, but not its C-terminus was able to interact with AR. Additional analysis suggested that TZF and TZF-L were able to form both homodimers and heterodimers. TZF-L inhibited the homodimer formation of TZF and the intranuclear dot formation of TZF. We propose that in the unique regulation system of AR-mediated transactivation, two spliced isoforms of TZF act as coactivator and corepressor, respectively.
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- 2005
30. 507 A Novel Mechanism of Fas Signaling Suppression by PMLRARα in Acute Promyelocytic Leukemia
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Rong-Hua Tao, Rezaeian Abdol Hossein, Felipe Samaniego, Zuzana Berkova, Li Bai, Jillian F. Wise, Urszula Daniluk, and Xue Ao
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Acute promyelocytic leukemia ,Cancer Research ,Promyelocytic leukemia protein ,Oncology ,biology ,Mechanism (biology) ,business.industry ,medicine ,biology.protein ,Cancer research ,Hematology ,medicine.disease ,business - Published
- 2011
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31. Regulation of Fas-mediated Apoptosis in B-Cell Lymphomas by Nucleolin
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Keyur P. Patel, Xue Ao, Jorge E. Romaguera, Sattva S. Neelapu, Wenzhuo Zhuang, Timothy J. McDonnell, Haifeng Zhu, Felipe Samaniego, Zuzana Berkova, Jillian F. Wise, Rohit Mathur, Frank K. Braun, Peter McLaughlin, Rong-Hua Tao, Luis Fayad, Larry W. Kwak, Zeming Chen, and Michael Wang
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Immunology ,Cell Biology ,Hematology ,Transfection ,Biology ,Fas receptor ,Biochemistry ,Molecular biology ,Fas ligand ,medicine.anatomical_structure ,Apoptosis ,Cell culture ,Cancer cell ,Cancer research ,medicine ,Nucleolin ,B cell - Abstract
Abstract 2406 The death receptor Fas has a key role in mediating homeostasis, elimination of defective cells and more recently promotion of cancer. Many effective anti-cancer therapies depend on Fas-mediated apoptosis to eradicate tumor cells and ineffective Fas- apoptotic signaling is a basis for primary as well as acquired resistance to chemotherapy. We hypothesized that Fas is subjected to direct regulation and inhibition of Fas attained by cancer cells and may explain the emergence of chemoresistance. To screen for potential binding modulators of Fas, we analyzed lymphoma cells for Fas binding proteins. We first purified Fas associated proteins by using activating CH-11 antibody bound to intact BJAB cells. After immunoprecipitation, any remaining Fas, considered activation–resistant, was subjected to the second immunoprecipitation with Fas antibody B-10 followed by liquid chromatography and tandem mass spectroscopy. This purification scheme identified high scoring peptides derived from nucleolin, a nuclear protein known to be overexpressed in cancer. Nucleolin is selectively expressed on the surfaces of cancer cells and blood vessels undergoing angiogenesis. In a cell culture system, we confirmed binding of nucleolin to Fas and the presence of nucleolin-Fas complexes on the surface of lymphoma cells by surface biotin labeling. Using deletion mutants of nucleolin, we identified RNA binding domain 4 and glycine/arginine rich region to be required for the binding to Fas. BJAB cells with partially knockdown (KD) nucleolin showed significantly higher rates of apoptosis in response to stimulation with CH-11 and FasL when compared to nontarget KD controls. Importantly, the lower levels of nucleolin in knockdown cells did not affect total and surface Fas expression. Nucleolin present on the cell surface prevented binding of FasL and CH-11 to the receptor and thus provides a mechanism for blocking activation of Fas apoptosis. To examine the role of nucleon in vivo, we transfected mice with nucleolin-expressing plasmids using the hydrodynamic transfection method. The mice overexpressing nucleolin showed significantly higher survival rates than vector control transfected mice (P=.01) after a challenge with a lethal dose of agonistic anti-Fas antibody. We next examined the expression of nucleolin in human lymphomas. Cell lines derived from lymphomas of different histological types consistently expressed nucleolin protein. We found nucleolin expressed on the surface of cells in over 20 primary lymphoma isolates, whereas peripheral blood lymphocytes showed low or undetectable levels. Lymphoma tissue microarray staining showed a correlation between nucleolin and Ki-67 expression. Whether nucleolin expression also correlates with adverse clinical features in lymphoma is currently under evaluation. Taken together, we show here that the known cancer associated protein nucleolin is overexpressed on surface of lymphoma cells where it binds to Fas receptor and blocks Fas signaling and apoptosis. We expect that further analysis of nucleolin properties will reveal how Fas-nucleolin interaction can be targeted to enhance killing of cancer cells leading eventually to cell surface nucleolin targeting therapy. Disclosures: Fayad: Roche: Research Funding.
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- 2012
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32. Mechanism of Regulating Fas Signaling by Promyelocytic Leukemia Protein (PML) and Its Dominant-Negative Mutant PMLRARα
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Zeming Chen, Zuzana Berkova, Wenzhuo Zhuang, Haifeng Zhu, Jillian F. Wise, Rohit Mathur, Frank K. Braun, Rong-Hua Tao, Felipe Samaniego, and Xue Ao
- Subjects
biology ,Chemistry ,Immunology ,Cell Biology ,Hematology ,Transfection ,Biochemistry ,Fas ligand ,Promyelocytic leukemia protein ,chemistry.chemical_compound ,Apoptosis ,Cancer cell ,biology.protein ,Cancer research ,Propidium iodide ,FADD ,Signal transduction - Abstract
Abstract 1327 Objective: Chemotherapies and irradiation depend on an intact Fas signaling system to eradicate cancer cells. Defective Fas signaling is an important cause of acquired resistance to cancer therapy. If we were able to restore Fas apoptosis or sensitize cancer cells to Fas-mediated apoptosis, we could improve the efficacy of many current cancer therapies. To elucidate defects of Fas signaling in cancer cells, we sought to identify potential modulators of Fas selectively expressed in cancers cells and target them to sensitize cancer cells to Fas-mediated apoptosis as a component of chemotherapy. Methods: Liquid chromatography tandem mass spectroscopy was used to identify Fas-associated proteins. Co-immunoprecipitation (co-IP) and Western Blot (WB) were used to detect/confirm interactions of PML and PMLRARα with Fas, and components of death-inducing signaling complex (DISC) FADD, c-FLIP, and caspase-8 cleavage in tissues from PML wild-type (WT) and knock-out (KO) mice and acute promyelocytic leukemia (APL) cells. Deletional mutagenesis was used to map protein interacting domains. PML shRNA lentivirus and As2O3 were used to downregulate PML and PMLRARα. Flow cytometry analysis of propidium iodide- and Annexin-V-stained cells was used to detect apoptosis in response to Fas stimulation. Mice transfected with PMLRARα were monitored for survival after a lethal challenge with agonistic Fas antibody Jo2 and tissues were analyzed for apoptosis by staining for cleaved caspase-3 and TUNEL. Results: The promyelocytic leukemia protein (PML) was identified as a Fas-binding protein by mass spectroscopy analysis. Using co-IP/WB analysis of tissues from PML WT and KO mice, we found PML interaction with Fas and FADD in PML WT MEF cells and liver cells but absent in KO MEF and liver cells; PML-Fas complexes were exclusively present in the membrane/cytoplasmic extracts but not in the nuclear extracts. The B-box domain of PML was found to be required for Fas binding. Knockdown of PML was associated with suppressed rates of Fas-mediated apoptosis compared to non-targeted knockdown cells; PML KO cells reconstituted with cytoplasmic PML were sensitized to Fas apoptosis. Furthermore, we found that liver cells from PML KO mouse showed impaired assembly of the Fas death-inducing signaling complex (DISC) in response to Fas activation when compared to PML WT. PML functions are known to be blocked by its dominant-negative form PMLRARα. We found PMLRARα interaction with Fas in primary human and transgenic mouse APL cells blocked Fas-mediated apoptosis. Blockage of apoptosis was mediated through PMLRARα -dependent recruitment of c-FLIPL/Sto and exclusion of procaspase-8 from the DISC. PMLRARα effects were also observed in vivo, as expression of PMLRARα protected mice against a lethal effect of agonistic anti-Fas antibody (P Conclusions: PML binds to Fas and promotes Fas-mediated apoptosis through enhancing Fas DISC formation while binding of PMLRARα to Fas blocks Fas-mediated apoptosis in APL by forming an apoptotic inhibitory complex enriched in c-FLIP. Our data suggest that PML plays a critical role in initiation of Fas signaling; in contrast, the dominant-negative mutant PMLRARα is a confirmed cancer specific inhibitor of Fas-mediated apoptosis. Thus, deficiency of PML or expression of PMLRARα can contribute to cancer development and resistance to chemotherapy. The newly discovered PML-Fas and PMLRARα -Fas complexes can be sites for modulation of apoptosis. Thus, by neutralizing the inhibitory effect of Fas-binding proteins such as PMLRARα and/or promoting positive Fas modulators such as PML, we can improve responses to chemotherapy that depend on activation of death receptors for effective elimination of cancer cells. Disclosures: No relevant conflicts of interest to declare.
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- 2012
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33. Abstract 4965: Oncoprotein PMLRARα directly suppresses Fas-mediated apoptosis through forming an apoptotic inhibitory complex with c-FLIP in vivo
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Celine Kerros, Jillian F. Wise, Xue Ao, Rong-Hua Tao, Zuzana Berkova, Haifeng Zhu, Felipe Samaniego, and Yong Seok Lee
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Acute promyelocytic leukemia ,Cancer Research ,Biology ,Fas receptor ,medicine.disease ,Molecular biology ,Fas ligand ,chemistry.chemical_compound ,Promyelocytic leukemia protein ,Oncology ,chemistry ,Apoptosis ,Cancer cell ,biology.protein ,medicine ,Propidium iodide ,Death domain - Abstract
Objective: Many genotoxic therapies, including radiation, depend on intact Fas signaling to eradicate cancer cells. Defective Fas signaling is an important cause of cancer resistance to therapy. Restoring Fas apoptosis or sensitizing cancer cells to Fas-mediated apoptosis would improve the efficacy of many cancer therapies. To elucidate a role for specific regulators of Fas signaling in cancer cells, we sought to identify potential modulators of Fas expressed in cancers and target them to selectively sensitize cancer cells to Fas-mediated apoptosis as a component of chemotherapy. Methods: Liquid chromatography tandem mass spectrometry was used to identify Fas-associated proteins; co-immunoprecipitation and Western blot were used to detect interactions of PMLRARα, PML, c-FLIP and Fas, and to examine the components of death-inducing signaling complex (DISC) and caspase-8 cleavage. Deletional mutagenesis was used to map the interaction domains. PML shRNA lentivirus and As2O3 were used to knock down PML and PMLRARα. Flow cytometry analysis of propidium iodide- and Annexin-V-stained cells was used to detect apoptosis. Mice were transfected with PMLRARα, monitored for survival, and tissues were analyzed for apoptosis by staining for cleaved caspase-3 and TUNEL. Results: We identified promyelocytic leukemia protein (PML) as a Fas-interacting protein using mass spectrometry analysis. The function of PML is blocked by its dominant-negative form PMLRARα. We found PMLRARα interaction with Fas in acute promyelocytic leukemia (APL)-derived cells and APL primary cells, and PML-Fas complexes in normal tissues. Binding of PMLRARα to Fas was mapped to the B-box domain of PML moiety and death domain of Fas. PMLRARα blockage of Fas apoptosis was demonstrated in U937/PR9 cells, human APL cells and transgenic mouse APL cells, in which PMLRARα recruited c-FLIPL/S and excluded procaspase-8 from Fas death signaling complex. PMLRARα expression in mice protected the mice against a lethal dose of agonistic anti-Fas antibody (P Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4965. doi:1538-7445.AM2012-4965
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- 2012
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34. PML and PMLRARα Interact with Fas to Regulate Fas-Mediated Apoptosis In Vivo
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Jeffrey J. Molldrem, Felipe Samaniego, Jillian F. Wise, Celine Kerros, Rong-Hua Tao, Hui Kuan Lin, David H. Hawke, Judith E. Karp, and Zuzana Berkova
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Acute promyelocytic leukemia ,biology ,HL60 ,Immunology ,Cell Biology ,Hematology ,Transfection ,Fas receptor ,medicine.disease ,Biochemistry ,Molecular biology ,Fas ligand ,Promyelocytic leukemia protein ,chemistry.chemical_compound ,chemistry ,Apoptosis ,Cancer cell ,medicine ,biology.protein - Abstract
Abstract 2451 Background: Altered Fas signaling leads to resistance to various anticancer therapies. The presence of at least low level expression of Fas is required for the transformed phenotype of many cells. In order for Fas to offer this complex regulation and to prevent inadvertent Fas activation, we propose that Fas signaling is subjected to tight regulation and this regulation could produce resistance to apoptosis of cancer cells. Methods: Cell lines NB4 (PMLRARα positive), HL60, U937/PR9 (PMLRARα expression driven by metalothionine promoter), or transfected HEK293 cells were collected and cell extracts were prepared for immunoprecipitation and Western blot analysis with the indicated antibodies. Cellular fractionation was performed using the nuclear/cytosol fractionation kit (BioVision, Mountain View, CA), according to the manufacturer's instructions. We expressed PMLRARα in C57B6 female mice by transfection of PMLRARα plasmid or empty-vector plasmid (12 mice per group) by tail vein inoculation using a hydrodynamic transfection method. Twenty-four hours after transfection, mice were challenged intraperitoneally with 2 μg/g mouse weight of agonistic Fas antibody Jo2, and monitored for survival. Liver tissues were stained with TUNEL assay and antibodies targeting apoptosis proteins. Differences between two groups were assessed using the 2-tailed student's t test and a P value < 0.05 was considered to be statistically significant. Results: In this study, we identified, by mass spectrometry analysis of Fas-associated proteins, the proapoptotic promyelocytic leukemia protein (PML) as a Fas-interacting protein. PML is a ubiquitously expressed tumor suppressor, which is downregulated or mutated in over 50% of all cancers. PML promotes apoptosis especially under conditions of cell stress. The function of PML is blocked by its dominant-negative form promyelocytic leukemia - retinoic acid receptor α (PMLRARα). Our focus on PMLRARα showed that it interacts with Fas in acute promyelocytic leukemia-derived cell lines and APL primary cells. Because PMLRARα is considered to be a nuclear transcription factor, we fractionated cell compartment proteins and identified PMLRAR α-Fas complexes present only in the cytoplasmic fraction. Binding of PML to Fas was mapped to the B-box domain of PML and death domain of Fas. PMLRARα blockage of apoptosis was shown in U937/PR9 and NB4 cells, in which PMLRARα recruited c-FLIPL/S and excluded procaspase 8 from the Fas death-inducing signaling complex. PMLRARα expression in mice protected the mice against a lethal dose of agonistic anti-Fas antibody (P Conclusion: Together, PMLRARα binds to Fas and blocks Fas-mediated apoptosis in APL through the accumulation of c-FLIP- Fas complexes. Furthermore, the common expression of PML-Fas complexes suggests Fas signaling is routinely regulated in the cytoplasm by PML, but susceptible to the blocking effects of PMLRARα in cancer. Disclosures: Karp: Schering Merck: Research Funding.
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- 2011
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35. Abstract 2908: Promyelocytic leukemia-retinoic acid receptor α forms complexes with Fas and FLIP and impairs Fas-mediated apoptosis
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Jillian F. Wise, Felipe Samaniego, Urszula Daniluk, Rong-Hua Tao, and Zuzana Berkova
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Fas mediated apoptosis ,Cancer Research ,Leukemia ,Retinoic acid receptor ,Oncology ,Biochemistry ,Chemistry ,Flip ,medicine ,Cancer research ,medicine.disease ,Fas receptor - Abstract
Fas plays a critical role in cell proliferation and in the selective killing of autoreactive lymphocytes and abnormal cells, including infected cells. To explain the common expression of Fas and the resistance to the Fas-induced apoptosis observed in some normal and cancer cells, we have screened cells for potential regulators of the Fas death receptor. By using mass spectroscopy analysis of Fas-associated proteins, we identified peptides derived from promyelocytic leukemia (PML). PML enhances pro-apoptotic signaling, while the promyelocytic leukemia-retinoic acid receptor α (PMLRARα) activates pro-survival pathways. Given these opposing functions, we tested whether PMLRARα, which typically operates in a dominant-negative manner, blocks Fas-mediated apoptosis. Co-immunoprecipitation analysis demonstrated that PMLRARα interacts with Fas in acute promyelocytic leukemia (APL)-derived NB4 cells, U937-PR9 cells and in APL primary cells. The binding of PMLRARα to Fas was mapped to the B-box domain of PMLRARα. Flow cytometry analysis of propidium iodide-stained and Annexin-V-stained cells challenged with Fas ligand (FasL) or agonistic anti-Fas antibody (CH-11) indicated that the presence of PMLRARα was associated with blocked Fas-mediated apoptosis at early and late stages. The knockdown of PMLRARα with shRNA sensitized the NB4 cells to Fas-mediated apoptosis. Expression of PMLRARα in U937-PR9 cells prevented Fas-mediated cleavage of procaspase-8 and also prevented procaspase-8 from binding to the Fas complex upon stimulation with the agonistic anti-Fas antibody (CH-11). Further analysis indicated that PMLRARα bound to FLIPL/S and forms a complex with Fas associated with suppression of Fas signaling. These data suggest that cancer-specific inhibitors of Fas such as PMLRARα block Fas-mediated apoptosis and thus can contribute to cancer development and resistance to therapy. Our results may provide an explanation for the long-known role of PMLRARα and PML in the regulation of Fas signaling, which we have shown to occur by direct regulation. We have identified an attractive potential target to the regulation of apoptosis at the PMLRARα-Fas and PML-Fas interfaces. By neutralizing the effect of death receptor inhibitors such as PMLRARα and other potential inhibitors, we can improve on the success of the many chemotherapeutic treatments that depend on activation of death receptors for effective elimination of cancer cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2908.
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- 2010
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36. Abstract 1254: Kaposi's Sarcoma herpesvirus K1's transcellular inhibition of Fas-mediated apoptosis
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Jillian F. Wise, Urszula Daniluk, Felipe Samaniego, Om Prakash, Zuzana Berkova, Rezaeian Abdol Hossein, Rong-Hua Tao, and Shu Wang
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Genetically modified mouse ,Cancer Research ,Pathology ,medicine.medical_specialty ,Lymphocyte ,Spleen ,Biology ,medicine.disease ,Fas receptor ,Molecular biology ,Lymphoid hyperplasia ,Lymphoma ,medicine.anatomical_structure ,Oncology ,Apoptosis ,medicine ,Lymph ,medicine.symptom - Abstract
The long-term expression of human herpesvirus 8 (HHV-8) K1 produces hyperplasia of lymph nodes, splenomegaly, and lymphomas in mice. The mechanism of how K1 causes hyperplasia and lymphomas is not known. K1 is known to activate Akt and nuclear factor kappa B (NF-kB) through immunoreceptor tyrosine-based activation motif (ITAM) and may also bind to Fas receptor through its immunoglobulin (Ig) chain-like domain and interfere with apoptosis. We thus hypothesized that development of hyperplasia and lymphomas in K1-expressing mice is driven by altered Fas signaling. Examination of mice expressing K1 via a ubiquitous promoter showed that 90% K1 transgenic mice (n=10) had developed lymphoid hyperplasia (at least 3 lymph nodes >3 mm) and 60% developed lymphomas after 18 months, while all (26) control nontransgenic mice remained free of lymph node hyperplasia, splenomegaly, and lymphoma. Some K1 mice developed liver or mesenteric tumors (4 of 10 mice). The spleens of 78% of K1 mice were enlarged at 18 months and were on average 3.5 times heavier than spleens of non-K1 transgenic control mice. Hematoxylin and eosin staining of spleen sections showed lymphocyte expansion in the periarteriolar lymphocyte sheath with disruption of normal spleen architecture. Anti-kappa and anti-lambda light chain antibodies revealed the presence of monoclonal foci in 3 out of 3 K1 mice (average 6 foci per single section of spleen), but no foci were present in 4 control non-transgenic mice. Moreover, K1 protein was expressed in approximately 10% of splenic cells after staining with anti-K1 antibody 2H5. In vitro overexpression of an Ig domain-containing protein CD79b or treatment cells with K1 peptides revealed competition with K1-Fas binding in a dose-dependent manner and rate enhancement of Fas-mediated apoptosis. We have also shown that K1 suppressed Fas-mediated apoptosis, even in cells that did not express K1. Transfection of K1 into one pool of mouse cells protected against Fas-mediated apoptosis of a second pool of human Fas-transfected mouse cells indicating protection in trans. This analysis indicates a key role of K1 in suppression of Fas-mediated apoptosis which operates in a cis and trans protective role against apoptosis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1254.
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- 2010
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37. Peptides Compete in Human Herpesvirus 8 K1-Fas Complexes Associated with Hyperplasia and Restore Apoptosis
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Zuzana Berkova, Felipe Samaniego, Steven Ovu, Om Prakash, Jillian F. Wise, Urszula Daniluk, and Rong-Hua Tao
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medicine.medical_specialty ,biology ,Lymphocyte ,Immunology ,Spleen ,Cell Biology ,Hematology ,Hyperplasia ,medicine.disease ,Biochemistry ,Molecular biology ,Lymphoid hyperplasia ,Lymphoma ,medicine.anatomical_structure ,Endocrinology ,Apoptosis ,Internal medicine ,biology.protein ,medicine ,Splenocyte ,Antibody ,medicine.symptom - Abstract
Abstract 2965 Poster Board II-941 The long-term expression of human herpesvirus 8 (HHV-8) K1 produces hyperplasia of lymph nodes, splenomegaly, and lymphomas in mice. The mechanism of how K1 causes hyperplasia and lymphomas. The immunoreceptor tyrosine-based activation motif (ITAM) of K1 was shown previously to activate of Akt and nuclear factor kappa B (NF-kB). However, we have recently shown that K1 suppresses Fas-mediated apoptosis through its extracellular immunoglobulin-like domain and that K1-transfected mice survive a lethal dose of agonistic anti-Fas antibody (Jo2). We thus hypothesized that development of hyperplasia and lymphomas in K1-expressing mice is driven by altered Fas signaling. Examination of mice expressing K1 via a ubiquitous promoter showed that K1 transgenic mice (n=10) had 90% developed lymphoid hyperplasia (at least 3 lymph nodes >3 mm) and 60% developed lymphomas after 18 months, while all (26) control nontransgenic mice remained free of lymph node hyperplasia, splenomegaly, and lymphoma. Some K1 mice developed liver or mesenteric tumors (4 of 10 mice). The spleens of 78% of K1 mice were enlarged at 18 months and were on average 3.5 times heavier than spleens of non-K1 transgenic control mice. Hematoxylin and eosin staining of spleen sections showed lymphocyte expansion in the periarteriolar lymphocyte sheath with disruption of normal spleen architecture. Anti-kappa and anti-lambda light chain antibodies revealed the presence of monoclonal foci in 3 out of 3 K1 mice (average 6 foci per single section of spleen), but no foci were present in 4 control non-transgenic mice. Moreover, K1 protein was expressed in approximately 10% of splenic cells after staining with anti-K1 antibody 2H5. To test the hypothesis that expression of K1 protein confers resistant to Fas-mediated apoptosis, splenic cells of 6-month-old K1 mice (n=3) and matched controls (n=3) were isolated and incubated with 50 ng/mL of agonistic anti-Fas antibody Jo2. At 12 hours of treatment, only 4±1% of splenocytes from K1 mice versus 17±2% of control splenocytes showed morphology of apoptosis (P We mapped the region that K1 uses to bind to Fas to an immunoglobulin (Ig) chain-like domain by expressing deletion mutants of K1. Overexpression of an Ig domain-containing protein CD79b competed with K1-Fas binding in a dose-dependent manner. Two 20-amino acid peptides (N251, N253) representing the Ig domain of K1 competed with K1-Fas binding in immunoprecipitation/immunoblotting analysis. The N251 and N253 peptides (100 mM) enhanced anti-Fas antibody (CH-11, 50 ng/mL)-induced apoptosis of BJAB lymphoma cells that expressed K1 but not that of vector-transfected BJAB cells. K1 splenocytes incubated with N251 and N253 showed enhanced rates of Fas-mediated apoptosis over control peptide-treated K1, and peptides did not enhance the apoptosis rates of control non-K1 expressing splenocytes. This analysis indicates a key role of K1 in lymphoid hyperplasia and lymphoma and that K1 direct binding to Fas occurs in vivo, which is effectively targeted by competing peptides to restore apoptosis. Disclosures: No relevant conflicts of interest to declare.
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- 2009
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38. Direct Inhibition of Fas Activation by CD74
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Jillian F. Wise, Urszula Daniluk, Rong-Hua Tao, Shu Wang, Zuzana Berkova, Michael Garcia, and Felipe Samaniego
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CD74 ,Immunology ,Cell Biology ,Hematology ,Biology ,Fas receptor ,Biochemistry ,Fas ligand ,Cell biology ,medicine.anatomical_structure ,Apoptosis ,Cancer cell ,medicine ,Hepatocyte growth factor ,Receptor ,B cell ,medicine.drug - Abstract
Abstract 3770 Poster Board III-706 Hematopoietic cancers are commonly resistant to Fas-mediated apoptosis that contributes to their resistance to chemotherapy and reversing Fas resistance has become a primary interest in treating chemotherapy-resistant hematopoietic cancers. Wide expression of Fas receptor yet the limited extent of Fas-mediated apoptosis in tissues suggest a strict regulation of Fas signaling. A recently identified tissue-specific modulator of Fas-mediated apoptosis is hepatocyte growth factor (HGF) receptor HGFR/Met. HGFR/Met binds Fas and inhibits Fas signaling at an immediate early step. However, upon binding of ligand HGF to HGFR/Met, Fas is released and Fas signaling is restored. We hypothesized that hematopoietic cancers may express similar inhibitors of Fas, that can be targeted to resensitize cancer cells to Fas and thus also to therapy. We screen lymphoma cells for for such inhibitors. B cell lymphoma-derived BJAB cells and primary patient samples were incubated with Fas agonistic antibody. Upon removal of antibody excess, cell lysates were immuno-depleted of CH-11 antibody-bound and thus activated Fas. The remaining supernatant was then precipitated a second time with anti-Fas antibody B-10 that binds to the intracellular region of Fas. Protein bands present in B-10 precipitates and absent in CH-11 precipitates were excised from silver-stained gel and analyzed by mass-spectroscopy. CD74, also termed the invariant chain of MHC II and known to mediate pro-survival signaling as a receptor for macrophage migration inhibitory factor (MIF), was found to exclusively associate with activation-resistant Fas. Intriguingly, CD74 is known to be overexpressed in 80% to 95% of hematopoietic cancers and also in some solid tumors. Using RNA interference we showed that CD74 negative cells are more sensitive to Fas-mediated apoptosis and that CD74 overexpression confers resistance to Fas both in vitro and in mouse tissues. Incubation of cells with peptides or anti-CD74 antibody LL1 disrupts the CD74-Fas inhibitory complex and sensitizes CD74-positive cells to Fas-mediated apoptosis. Analysis at the molecular level revealed that CD74 interferes with the immediate early steps in Fas signaling – binding of agonistic CH-11 antibody and subsequent clustering of Fas receptor. We anticipate that specific disruption of the CD74-Fas interaction by CD74-derived peptides or anti-CD74 antibodies will sensitize cancer cells to Fas-mediated apoptosis. This approach represents a novel strategy for modulation of cell surface death receptors to produce effective treatments for CD74-positive cancers. Disclosures: No relevant conflicts of interest to declare.
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- 2009
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39. Promyelocytic Leukemia-Retinoic Acid Receptor α Binds and Prevents Activation of the Fas Receptor and Suppresses Fas-Mediated Apoptosis
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Jillian F. Wise, Zuzana Berkova, Rong-Hua Tao, Urszula Daniluk, and Felipe Samaniego
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Acute promyelocytic leukemia ,Chemistry ,Immunology ,Cell Biology ,Hematology ,medicine.disease ,Fas receptor ,Biochemistry ,Fas ligand ,Retinoic acid receptor ,Leukemia ,Apoptosis ,Cancer cell ,medicine ,Cancer research ,Receptor - Abstract
Abstract 3976 Poster Board III-912 Fas plays a critical role in cell proliferation and in the selective killing of autoreactive lymphocytes and abnormal cells, including infected cells. To explain the common expression of Fas and the resistance to the Fas killing observed in some normal and cancer cells, we have screened cells for potential regulators of the Fas death receptor. By using mass spectroscopy analysis of Fas-associated proteins, we identified a group of peptides derived from promyelocytic leukemia (PML). PML enhances pro-apoptotic signaling, while the promyelocytic leukemia–retinoic acid receptor α (PMLRARα) activates pro-survival pathways. Given these opposing functions, we tested whether PMLRARα, which typically operates in a dominant-negative manner, blocks Fas-mediated apoptosis. Co-immunoprecipitation analysis demonstrated that PMLRARα interacts with Fas in acute promyelocytic leukemia (APL)-derived NB4 cells, U937-PR9 cells and in APL primary cells. The binding of PMLRARα to Fas was mapped to the B-box domain of PMLRARα. Flow cytometry analysis of propidium iodide-stained and Annexin-V-stained cells challenged with Fas ligand (FasL) or agonistic anti-Fas antibody (CH-11) indicated that the presence of PMLRARα was associated with blocked Fas-mediated apoptosis at early and late stages. The knockdown of PMLRARα with shRNA sensitized the NB4 cells to Fas-mediated apoptosis. Expression of PMLRARα in U937-PR9 cells prevented Fas-mediated cleavage of procaspase-8 and also prevented procaspase-8 from binding to the Fas complex upon stimulation with the agonistic anti-Fas antibody (CH-11). Further analysis indicated that PMLRARα bound to FLIPL/S and forms an apoptotic inhibitory complex with Fas, which prevents Fas activation. The data suggest that tissue-specific inhibitors of Fas such as PMLRARα block Fas-mediated apoptosis and thus can contribute to cancer development. Our results may provide an explanation for the long-known role of PMLRARα and PML in the regulation of Fas signaling, which we have shown to occur by direct regulation. We have identified an attractive potential target to the regulation of apoptosis at the PMLRARα-Fas and PML-Fas interfaces. By neutralizing the effect of death receptor inhibitors such as PMLRARα and other potential inhibitors, we can improve on the success of the many chemotherapeutic treatments that depend on activation of death receptors for effective elimination of cancer cells. Disclosures: No relevant conflicts of interest to declare.
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- 2009
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40. CD74 Is a Regulator of Fas-Mediated Apoptotic Signaling
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Suizhao Wang, Hoyoung Maeng, Jillian F. Wise, David H. Hawke, Rong-Hua Tao, Zuzana Berkova, Shu Wang, Felipe Samaniego, and Larry W. Kwak
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CD74 ,biology ,Chemistry ,Immunology ,Cell Biology ,Hematology ,Transfection ,medicine.disease_cause ,Immunoglobulin light chain ,Biochemistry ,Haematopoiesis ,Cell culture ,Apoptosis ,Cancer research ,biology.protein ,medicine ,Antibody ,Carcinogenesis - Abstract
Resistance to Fas-mediated apoptosis in hematopoietic cancers interferes with the efficacy of currently available chemotherapy. Our prior studies of human herpesvirus 8 oncoprotein K1 showed that K1 binds to Fas and interferes with activation of Fas-mediated apoptotic signaling (Wang, W, et al, Blood2007; 109:5455–62). Herpesvirus proteins often mimic host proteins or their functions, we thus searched for cellular proteins associated with inactive Fas in order to identify potential regulators of Fas signaling. We identified CD74, the invariant light chain of the major histocompatibility class II complex, associated exclusively with inactive/activation resistant Fas in B-cell lymphoma-derived BJAB cell line. Interestingly, overexpression of CD74 has been previously reported in ~ 90% of hematopoietic cancers and derived cell lines (Stein, R, et al., Ciln Cancer Res.2007; 13: 5556s–63s), as well as in ~ 80% of non-small-cell lung carcinoma (Ioachim, H.L., Am J Surg Pathol.1996; 20: 64–71). The role of CD74 in carcinogenesis was thus suspected. Through siRNA suppression of CD74 expression in BJAB cells we have determined that CD74 positive cells are more resistant to agonistic antibody CH-11-induced Fas-mediated apoptosis then the CD74 siRNA transfected cells (38 ± 7.8 % vs. 54 ± 9.8 %; P = 0.045). In addition, a challenge with agonistic anti-Fas antibody showed a significant survival advantage of the mice expressing CD74 in their livers over the vector-transfected mice (83% vs. 0 %; P < 0.016). We have mapped domain of CD74 required for its association with Fas and for significant protection of mice to a membrane-proximal extracellular region of CD74. Treatment of BJAB cells for 24 hours with humanized anti-CD74 antibody (hLL1 + crosslinking antibody), FasL-Fc, or anti-Fas antibody CH-11 induced apoptosis in 19%, 25% and 13% of cells, respectively. Combination of hLL1+crosslinking antibody with FasL-Fc or CH-11 increased apoptosis to ~ 45% of cells in both cases (P < 0.01), while combination of FasL-Fc or CH-11 with hLL1 + irrelevant antibody had no significant effect on the extent of apoptosis. Our results support the idea of an endogenous regulatory system of Fas-mediated apoptosis that utilizes transmembrane proteins interacting with Fas. We anticipate that specific blocking of the CD74-Fas interaction will sensitize CD74 overexpressing cancer cells to Fas-mediated apoptosis and thus will lead to a more effective chemotherapy for hematopoietic cancers.
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- 2008
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41. Human Herpesvirus 8 Protein K1 Interferes with Fas-Mediated Apoptosis Induces Clonal Growth and Lymphoid Hyperplasia
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Hoyoung Maeng, Zuzana Berkova, Rong-Hua Tao, Jillian F. Wise, Shu Wang, Suizhao Wang, and Felipe Samaniego
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Genetically modified mouse ,biology ,Immunology ,Lethal dose ,Cell Biology ,Hematology ,Hyperplasia ,medicine.disease ,Biochemistry ,Lymphoid hyperplasia ,Lymphoma ,Apoptosis ,medicine ,biology.protein ,Cancer research ,Lymph ,Antibody ,medicine.symptom - Abstract
Background: Expression of Human herpesvirus 8 (HHV-8) K1 causes hyperplasia of lymph nodes and lymphomas in mice. The exact mechanism of how K1 causes hyperplasia and lymphomas in K1 expressing mice is not known. The cytoplasmic immunoreceptor tyrosine-based activation motif (ITAM) of K1 was shown previously to be involved in activation of Nuclear Factor kappa B (NF-k-B). Moreover, we had shown recently that K1 suppresses Fas-mediated apoptosis through its extracellular immunoglobulin-like domain and that K1-transfected mice survived a lethal dose of agonistic anti-Fas antibody (Jo2). We thus hypothesized that development of hyperplasia and lymphomas in K1-expressing mice is driven by alterations of Fas signaling. Results: At 18 months, 10 K1 transgenic mice, 90% developed lymphoid hyperplasia (>3mm) and 60% developed lymphomas, while all (26) control mice remained hyperplasia and lymphoma free. In the extreme cases, K1 mice developed liver or mesenteric tumors (4 and 4 of 10 mice, respectively). Spleens of 78% of K1 mice were enlarged at 18 months and were on average 3.5 times heavier than spleens of non-expressing control mice (332 ± 200 mg vs. 94 ± 26 mg, P < 0.03). The H and E staining of spleen sections showed expansion to the periarteriolar lymphocyte sheath with disruption of normal follicular architecture. Staining of spleen sections with anti-kappa and anti-lambda light chain antibodies revealed presence of monoclonal foci in 3 out of 3 K1 mice (average 6 foci per single section of spleen), but none in the 4 control mice. Moreover, K1 protein was expressed in about 10% of splenic cells as judged from staining with anti-K1 antibody 2H5. To test the hypothesis that expression of K1 protein in spleens makes them resistant to Fas-mediated apoptosis, splenic cells of 6 month old K1 mice (n=3) and matched controls (n=3) were isolated and incubated with 50 ng/mL of agonistic anti-Fas antibody Jo2. At 12 hours of treatment, only 4 ± 1% of splenocytes from K1 mice versus 17 ± 2% of control splenocytes were undergoing apoptosis (P Conclusion: These results confirm that K1 is associated with lymphoid hyperplasia and lymphoma and provide plausible explanation. K1 blocks Fas-mediated apoptosis and competing peptides can reinstate apoptosis.
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- 2008
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42. Human Herpesvirus 8 Protein K1 Interference with Fas-Mediated Apoptosis Induces Clonal Growth Which Leads to Lymphoid Hyperplasia
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Hoyoung Maeng, Jillian F. Wise, Zuzana Berkova, Rong-Hua Tao, Suizhao Wang, and Shu Wang
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Pathology ,medicine.medical_specialty ,Immunology ,Cell Biology ,Hematology ,Biology ,Hyperplasia ,medicine.disease ,Biochemistry ,Lymphoid hyperplasia ,Lymphoma ,medicine.anatomical_structure ,Apoptosis ,medicine ,Mesenteric lymph nodes ,Lymph ,Primary effusion lymphoma ,medicine.symptom ,Kaposi's sarcoma - Abstract
Background: Human herpesvirus 8 (HHV-8) infection is causally associated with the development of primary effusion lymphoma and Kaposi s sarcoma. A transmembrane protein of HHV-8, K1, is readily expressed in those tumors and the expression of K1 alone causes hyperplasia of lymph nodes and lymphomas in mice. The exact mechanism of how K1 causes hyperplasia and lymphomas in K1 expressing mice is not known. The cytoplasmic immunoreceptor tyrosine-based activation motif (ITAM) of K1 was shown previously to be involved in activation of Nuclear Factor kappa B (NF -B). Moreover, we had shown recently that K1 suppresses Fas-mediated apoptosis through its extracellular immunoglobulin-like domain and that K1-transfected mice survived a lethal dose of agonistic anti-Fas antibody (Jo2). We thus hypothesized that development of hyperplasia and lymphomas in K1-expressing mice is driven by alterations of Fas signaling. Results: Gross examination of thoracic and abdominal cavities of transgenic mice with K1 expression driven by a ubiquitous promoter were sacrificed at 18 months of age revealed enlarged cervical, mediastinal, renal, mesenteric lymph nodes and spleens. Peyer s patches were also enlarged and readily visible on the outer surface of the ileum. Of 10 K1 mice, 90% developed lymphoid hyperplasia (>3mm) and 60% developed lymphomas, while all (26) control mice remained hyperplasia and lymphoma free. In the extreme cases, K1 mice developed liver or mesenteric tumors (4 and 4 of 10 mice, respectively). Spleens of 78% of K1 mice were enlarged at 18 months and were on average 3.5 times heavier than spleens of non-expressing control mice (332 200 mg vs. 94 26 mg, P < 0.03). The H and E staining of spleen sections showed expansion to the periarteriolar lymphocyte sheath with disruption of normal follicular architecture. Staining of spleen sections with antikappa and anti-lambda light chain antibodies revealed presence of monoclonal foci in 3 out of 3 K1 mice (average 6 foci per single section of spleen), but none in the 4 control mice. Moreover, K1 protein was expressed in about 10% of splenic cells as judged from staining with anti-K1 antibody 2H5. To test the hypothesis that expression of K1 protein in spleens makes them resistant to Fas-mediated apoptosis, splenic cells of 6 month old K1 mice (n=3) and matched controls (n=3) were isolated and incubated with 50 ng/mL of agonistic anti-Fas antibody Jo2. At 12 hours of treatment, only 4 1% of splenocytes from K1 mice versus 17 2% of control splenocytes were undergoing apoptosis (P Conclusion: These results over all confirm that K1 is associated with lymphoid hyperplasia and lymphoma and provide plausible explanation of this phenomenon. Interference of K1 with Fas-mediated apoptosis disrupts the normal life cycle of lymphocytes.
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- 2008
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43. The peptide derived from the Ig-like domain of human herpesvirus 8 K1 protein induces death in hematological cancer cells.
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Daniluk, Urszula, Kerros, Celine, Rong-Hua Tao, Wise, Jillian F., Xue Ao, Berkova, Zuzana, and Samaniego, Felipe
- Abstract
Background: Although significant progress has been made in the treatment of lymphomas, many lymphomas exhibit resistance to cell death, suggesting a defective Fas signaling, which remains poorly understood. We previously reported that cells expressing the K1 protein of human herpesvirus 8 (HHV-8) resist death through the complex formation of the Ig-like domain of K1 with Fas. Recently, we investigated whether peptides derived from the Ig-like domain of the K1 protein may affect cell death. Methods: K1 positive and negative cell lines were incubated with the K1-derived peptides, and cell death (apoptotic and necrotic) was assessed by flow cytometry and LDH assay. Activation of caspases was assessed by fluorometric assay and flow cytometry. Fas receptor-independent, peptide-mediated cell killing was tested in the Fas-resistant Daudi cell line and Jurkat cell clones deficient in caspase-8 and FADD functionality. Activation of TNF receptors I and II was blocked by pre-incubation with corresponding blocking antibodies. The effect of the K1 peptide in vivo was tested in a mouse xenograft model. Results: We observed that the peptide S20-3 enhanced cell death in K1-positive BJAB cells and HHV-8 positive primary effusion lymphoma (PEL) cell lines. Similar effects of this peptide were observed in B-cell lymphoma and T-lymphoblastic leukemia cells without K1 expression but not in normal human peripheral blood mononuclear cells. A single intratumoral injection of the S20-3 peptide decreased the growth of Jurkat xenografts in SCID mice. The mechanism of tumor cell death induced by the S20-3 peptide was associated with activation of caspases, but this activity was only partially inhibited by the pan-caspase inhibitor z-VAD. Furthermore, the K1 peptide also killed Fas-resistant Daudi cells, and this killing effect was inhibited by pre-incubation of cells with antibodies blocking TNFRI. Conclusion: Taken together, these findings indicate that the S20-3 peptide can selectively induce the death of malignant hematological cell lines by Fas- and/or TNFRI-dependent mechanisms, suggesting the K1-derived peptide or peptidomimetic may have promising therapeutic potential for the treatment of hematological cancers. [ABSTRACT FROM AUTHOR]
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- 2012
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44. Direct Suppression of Fas-Mediated Apoptosis by PMLRARa through Forming An Apoptotic Inhibitory Complex with c-FLIP In Acute Promyelocytic Leukemia
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Felipe Samaniego, Jillian F. Wise, Zuzana Berkova, Rong-Hua Tao, Daniluk Urszula, Xue Ao, and Li Bai
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Acute promyelocytic leukemia ,Immunology ,Cell Biology ,Hematology ,Transfection ,Biology ,medicine.disease ,Fas receptor ,Biochemistry ,Fas ligand ,Cell biology ,chemistry.chemical_compound ,chemistry ,Apoptosis ,Cancer cell ,medicine ,Propidium iodide ,Death domain - Abstract
Abstract 3148 Fas plays a critical role in cell proliferation and in the selective killing of autoreactive lymphocytes and abnormal cells, including infected cells. To explain the common expression of Fas and the resistance to the Fas-induced apoptosis observed in some normal and cancer cells, we screened cells for potential regulators of the Fas death receptor. By using mass spectroscopy analysis of Fas-associated proteins, we identified peptides derived from promyelocytic leukemia (PML). PML enhances pro-apoptotic signaling, while its dominant negative form, promyelocytic leukemia–retinoic acid receptor α (PMLRARα) fusion protein, activates pro-survival pathways. Given these opposing functions, we tested whether PMLRARα blocks Fas-mediated apoptosis. Co-immunoprecipitation analysis demonstrated that PMLRARα interacts with Fas in acute promyelocytic leukemia (APL)-derived NB4 cells, U937/PR9 cells and APL primary cells isolated from patients. The PMLRARα-Fas binding was mapped to the PML B-box domain of PMLRARα and death domain of Fas. Flow cytometry analysis of propidium iodide- and Annexin V-stained cells challenged with Fas ligand (FasL) or agonistic anti-Fas antibody CH-11 indicated that PMLRARα blocks Fas-mediated apoptosis at early and late stages. In line with this finding, knockdown of PMLRARα with shRNA sensitized the NB4 cells to Fas-mediated apoptosis. Detailed analysis showed that expression of PMLRARα prevents procaspase-8 from binding to the Fas complex upon stimulation with the agonistic anti-Fas antibody (CH-11) and thus, also prevents cleavage/activation of procaspase-8. Further analysis indicated that PMLRARα recruits caspase-8 inhibitor c-FLIPL/S to Fas to suppress Fas signaling. A significantly higher number of mice transfected with PMLRARα-expressing plasmid than mice transfected with empty vector survived the treatment with the mouse agonistic anti-Fas antibody Jo2 (11 of 12 vs. 0 of 12; P < 0.001). Livers from PMLRARα-transfected mice contained fewer cleaved caspase-3 positive/apoptotic cells when compared with vector-transfected mice. These data suggest that PMLRARα is a cancer specific Fas-binding inhibitor of Fas-mediated apoptosis and thus, can contribute to cancer development and resistance to therapy. Our results may provide an explanation for the long-known role of PMLRARα and PML in the regulation of Fas signaling, which, as we have shown, can occur by regulation via direct interaction. The newly-discovered PMLRARα-Fas and PML-Fas complexes can be sites for modulation of apoptossis. By neutralizing the effect of death receptor inhibitors such as PMLRARα, we can improve responses to many chemotherapeutic treatments that depend on activation of death receptors for effective elimination of cancer cells. Disclosures: No relevant conflicts of interest to declare.
45. The peptide derived from the Ig-like domain of human herpesvirus 8 K1 protein induces death in hematological cancer cells
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Jillian F. Wise, Celine Kerros, Urszula Daniluk, Felipe Samaniego, Rong-Hua Tao, Xue Ao, and Zuzana Berkova
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Cancer Research ,Programmed cell death ,Fas-Associated Death Domain Protein ,Apoptosis ,Mice, SCID ,Jurkat cells ,lcsh:RC254-282 ,Receptors, Tumor Necrosis Factor ,03 medical and health sciences ,Jurkat Cells ,Mice ,Viral Proteins ,0302 clinical medicine ,Animals ,Humans ,FADD ,fas Receptor ,Caspase ,030304 developmental biology ,0303 health sciences ,Caspase 8 ,biology ,Research ,Fas receptor ,lcsh:Neoplasms. Tumors. Oncology. Including cancer and carcinogens ,Molecular biology ,3. Good health ,Cell biology ,Protein Structure, Tertiary ,Gene Expression Regulation, Neoplastic ,Cell killing ,Oncology ,030220 oncology & carcinogenesis ,Hematologic Neoplasms ,Cancer cell ,Herpesvirus 8, Human ,biology.protein ,Peptides ,Signal Transduction - Abstract
Background Although significant progress has been made in the treatment of lymphomas, many lymphomas exhibit resistance to cell death, suggesting a defective Fas signaling, which remains poorly understood. We previously reported that cells expressing the K1 protein of human herpesvirus 8 (HHV-8) resist death through the complex formation of the Ig-like domain of K1 with Fas. Recently, we investigated whether peptides derived from the Ig-like domain of the K1 protein may affect cell death. Methods K1 positive and negative cell lines were incubated with the K1-derived peptides, and cell death (apoptotic and necrotic) was assessed by flow cytometry and LDH assay. Activation of caspases was assessed by fluorometric assay and flow cytometry. Fas receptor-independent, peptide-mediated cell killing was tested in the Fas-resistant Daudi cell line and Jurkat cell clones deficient in caspase-8 and FADD functionality. Activation of TNF receptors I and II was blocked by pre-incubation with corresponding blocking antibodies. The effect of the K1 peptide in vivo was tested in a mouse xenograft model. Results We observed that the peptide S20-3 enhanced cell death in K1-positive BJAB cells and HHV-8 positive primary effusion lymphoma (PEL) cell lines. Similar effects of this peptide were observed in B-cell lymphoma and T-lymphoblastic leukemia cells without K1 expression but not in normal human peripheral blood mononuclear cells. A single intratumoral injection of the S20-3 peptide decreased the growth of Jurkat xenografts in SCID mice. The mechanism of tumor cell death induced by the S20-3 peptide was associated with activation of caspases, but this activity was only partially inhibited by the pan-caspase inhibitor z-VAD. Furthermore, the K1 peptide also killed Fas-resistant Daudi cells, and this killing effect was inhibited by pre-incubation of cells with antibodies blocking TNFRI. Conclusion Taken together, these findings indicate that the S20-3 peptide can selectively induce the death of malignant hematological cell lines by Fas- and/or TNFRI-dependent mechanisms, suggesting the K1-derived peptide or peptidomimetic may have promising therapeutic potential for the treatment of hematological cancers.
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46. Nucleolin inhibits Fas ligand binding and suppresses Fas-mediated apoptosis in vivo via a surface nucleolin-Fas complex.
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Wise, Jillian F., Berkova, Zuzana, Mathur, Rohit, Haifeng Zhu, Braun, Frank K., Rong-Hua Tao, Sabichi, Anita L., Xue Ao, Hoyoung Maeng, and Samaniego, Felipe
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NUCLEOLIN , *APOPTOSIS , *LYMPHOMAS , *B cell lymphoma , *BLOOD donors , *MICE , *IMMUNOGLOBULINS - Abstract
Resistance to Fas-mediated apoptosis is associated with poor cancer outcomes and chemoresistance. To elucidate potential mechanisms of defective Fas signaling, we screened primary lymphoma cell extracts for Fas-associated proteins that would have the potential to regulate Fas signaling. An activation-resistant Fas complex selectively included nucleolin. We confirmed the presence of nucleolin-Fas complexes in B-cell lymphoma cells and primary tissues, and the absence of such complexes in B-lymphocytes from healthy donors. RNA-binding domain 4 and the glycine/arginine-rich domain of nucleolin were essential for its association with Fas. Nucleolin colocalized with Fas on the surface of B-cell lymphoma cells. Nucleolin knockdown sensitized BJAB cells to Fas ligand (FasL)-induced and Fas agonistic antibody-induced apoptosis through enhanced binding, suggesting that nucleolin blocks the FasL-Fas interaction. Mice transfected with nucleolin were protected from the lethal effects of agonistic anti-mouse Fas antibody (Jo2) and had lower rates of hepatocyte apoptosis, compared with vector and a non-Fas-binding mutant of nucleolin. Our results show that cell surface nucleolin binds Fas, inhibits ligand binding, and thus prevents induction of Fas-mediated, apoptosis in B-cell lymphomas and may serve as a new therapeutic target. [ABSTRACT FROM AUTHOR]
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- 2013
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