Publications by authors named "Youzhong Yuan"

28 Publications

  • Page 1 of 1

Limitations of an ex vivo breast cancer model for studying the mechanism of action of the anticancer drug paclitaxel.

Eur J Pharmacol 2021 Jan 30;891:173780. Epub 2020 Nov 30.

Department of Biochemistry and Molecular Biology, University of Arkansas for Medical Sciences, Little Rock, AR, 72205, USA. Electronic address:

Paclitaxel is widely used in the treatment of breast, ovarian, lung, and other cancers. Its primary mechanism is to prevent microtubule depolymerization causing loss of dynamic instability crucial for normal microtubule function leading to mitotic arrest. Prolonged mitotic arrest results in cell death as a secondary response. The effects of paclitaxel are typically studied in cell lines which precludes assessment of the possible influence of tumor-associated cells. We therefore examined paclitaxel action ex vivo in fresh explant cultures of human breast tumors. Surprisingly, we found that paclitaxel failed to induce tumor cell death in explant culture, in contrast to several other cytotoxic agents including salinomycin and vincristine. The lack of effect was not due to defective drug uptake, and furthermore, analysis of H&E stained tumor slices indicated that paclitaxel treatment caused defective (granular) mitosis and chromosomal condensation in 5-10% of tumor cells after 72 h. These results suggest that while paclitaxel was able to penetrate into the tumor slice and disrupt mitosis in cycling tumor cells, any ensuing cell death likely occurred beyond the useful lifetime of the tumor slices. We conclude that explant culture systems may be inappropriate for the study of cytotoxic drugs where a delay exists between the drug's primary and secondary modes of action.
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http://dx.doi.org/10.1016/j.ejphar.2020.173780DOI Listing
January 2021

The Role of Sirtuin 3 in Radiation-Induced Long-Term Persistent Liver Injury.

Antioxidants (Basel) 2020 May 11;9(5). Epub 2020 May 11.

Division of Radiation Health, University of Arkansas for Medical Sciences, 4301 W. Markham St., Little Rock, AR 72205, USA.

In patients with abdominal region cancers, ionizing radiation (IR)-induced long-term liver injury is a major limiting factor in the use of radiotherapy. Previously, the major mitochondrial deacetylase, sirtuin 3 (SIRT3), has been implicated to play an important role in the development of acute liver injury after total body irradiation but no studies to date have examined the role of SIRT3 in liver's chronic response to radiation. In the current study, ten-month-old Sirt3 and Sirt3 male mice received 24 Gy radiation targeted to liver. Six months after exposure, irradiated Sirt3 mice livers demonstrated histopathological elevations in inflammatory infiltration, the loss of mature bile ducts and higher DNA damage (TUNEL) as well as protein oxidation (3-nitrotyrosine). In addition, increased expression of inflammatory chemokines (IL-6, IL-1β, TGF-β) and fibrotic factors (Procollagen 1, α-SMA) were also measured in Sirt3 mice following 24 Gy IR. The alterations measured in enzymatic activities of catalase, glutathione peroxidase, and glutathione reductase in the livers of irradiated Sirt3 mice also implied that hydrogen peroxide and hydroperoxide sensitive signaling cascades in the absence of SIRT3 might contribute to the IR-induced long-term liver injury.
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http://dx.doi.org/10.3390/antiox9050409DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7278565PMC
May 2020

Carbamate derivatives of colchicine show potent activity towards primary acute lymphoblastic leukemia and primary breast cancer cells-in vitro and ex vivo study.

J Biochem Mol Toxicol 2020 Jun 5;34(6):e22487. Epub 2020 Mar 5.

Department of Biochemistry and Molecular Biology, University of Arkansas for Medical Sciences, Little Rock, Arkansas.

Colchicine (COL) shows strong anticancer activity but due to its toxicity towards normal cells its wider application is limited. To address this issue, a library of 17 novel COL derivatives, namely N-carbamates of N-deacetyl-4-(bromo/chloro/iodo)thiocolchicine, has been tested against two types of primary cancer cells. These included acute lymphoblastic leukemia (ALL) and human breast cancer (BC) derived from two different tumor subtypes, ER+ invasive ductal carcinoma grade III (IDCG3) and metastatic carcinoma (MC). Four novel COL derivatives showed higher anti-proliferative activity than COL (IC  = 8.6 nM) towards primary ALL cells in cell viability assays (IC range of 1.1-6.4 nM), and several were more potent towards primary IDCG3 (IC range of 0.1 to 10.3 nM) or MC (IC range of 2.3-9.1 nM) compared to COL (IC of 11.1 and 11.7 nM, respectively). In addition, several derivatives were selectively active toward primary breast cancer cells compared to normal breast epithelial cells. The most promising derivatives were subsequently tested against the NCI panel of 60 human cancer cell lines and seven derivatives were more potent than COL against leukemia, non-small-cell lung, colon, CNS and prostate cancers. Finally, COL and two of the most active derivatives were shown to be effective in killing BC cells when tested ex vivo using fresh human breast tumor explants. The present findings indicate that the select COL derivatives constitute promising lead compounds targeting specific types of cancer.
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http://dx.doi.org/10.1002/jbt.22487DOI Listing
June 2020

Identification of a novel monocytic phenotype in Classic Hodgkin Lymphoma tumor microenvironment.

PLoS One 2019 12;14(11):e0224621. Epub 2019 Nov 12.

Department of Pathology, University of Arkansas for Medical Sciences, Little Rock, Arkansas, United States of America.

Classic Hodgkin lymphoma (CHL) characteristically shows few malignant cells in a microenvironment comprised of mixed inflammatory cells. Although CHL is associated with a high cure rate, recent studies have associated poor prognosis with absolute monocyte count in peripheral blood and increased monocyte/macrophages in involved lymph nodes. Thus, the role of monocytic infiltration and macrophage differentiation in the tumor microenvironment of CHL may be more relevant than absolute macrophage numbers to defining prognosis in CHL patients and potentially have therapeutic implications. Most studies identify tumor-associated macrophages (TAMs) using markers (e.g., CD68) expressed by macrophages and other mononuclear phagocytes, such as monocytes. In contrast, Class A Scavenger Receptor (SR-A/CD204) is expressed by tissue macrophages but not monocytic precursors. In this study, we examined SR-A expression in CHL (n = 43), and compared its expression with that of other macrophage markers. We confirmed a high prevalence of mononuclear cells that stained with CD68, CD163, and CD14 in CHL lymph nodes. However, SR-A protein expression determined by immunohistochemistry was limited to macrophages localized in sclerotic bands characteristic of nodular sclerosis CHL. In contrast, SR-A protein was readily detectable in lymph nodes with metastatic tumor, extra-nodal CHL, T cell/histiocyte-rich large B cell lymphoma, and resident macrophages in non-malignant tissues, including spleen, lymph node, liver and lung. The results of SR-A protein expression paralleled the expression of SR-A mRNA determined by quantitative RT-PCR. These data provide evidence that tumor-infiltrating monocyte/macrophages in CHL have a unique phenotype that likely depends on the microenvironment of nodal CHL.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0224621PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6850552PMC
March 2020

Coactivation of NF-κB and Notch signaling is sufficient to induce B-cell transformation and enables B-myeloid conversion.

Blood 2020 01;135(2):108-120

Department of Pathology, Carver College of Medicine, University of Iowa, Iowa City, IA.

NF-κB and Notch signaling can be simultaneously activated in a variety of B-cell lymphomas. Patients with B-cell lymphoma occasionally develop clonally related myeloid tumors with poor prognosis. Whether concurrent activation of both pathways is sufficient to induce B-cell transformation and whether the signaling initiates B-myeloid conversion in a pathological context are largely unknown. Here, we provide genetic evidence that concurrent activation of NF-κB and Notch signaling in committed B cells is sufficient to induce B-cell lymphomatous transformation and primes common progenitor cells to convert to myeloid lineage through dedifferentiation, not transdifferentiation. Intriguingly, the converted myeloid cells can further transform, albeit at low frequency, into myeloid leukemia. Mechanistically, coactivation of NF-κB and Notch signaling endows committed B cells with the ability to self renew. Downregulation of BACH2, a lymphoma and myeloid gene suppressor, but not upregulation of CEBPα and/or downregulation of B-cell transcription factors, is an early event in both B-cell transformation and myeloid conversion. Interestingly, a DNA hypomethylating drug not only effectively eliminated the converted myeloid leukemia cells, but also restored the expression of green fluorescent protein, which had been lost in converted myeloid leukemia cells. Collectively, our results suggest that targeting NF-κB and Notch signaling will not only improve lymphoma treatment, but also prevent the lymphoma-to-myeloid tumor conversion. Importantly, DNA hypomethylating drugs might efficiently treat these converted myeloid neoplasms.
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http://dx.doi.org/10.1182/blood.2019001438DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6952829PMC
January 2020

The response of phyllodes tumor of the breast to anticancer therapy: An and study.

Oncol Lett 2019 Nov 6;18(5):5097-5106. Epub 2019 Sep 6.

Department of Biochemistry and Molecular Biology, University of Arkansas for Medical Sciences, Little Rock, AR 72205, USA.

Phyllodes tumors of the breast (PTB) are uncommon stromal-epithelial neoplasms, with the main recommended treatment being surgical removal. However, even with adequate resection, the risk of recurrence in the malignant form remains as high as 40%, and there is no recognized consensus on the most effective drugs for PTB. In the present study, an model of malignant phyllodes and derived primary cell cultures were used to evaluate the effectiveness of a panel of different drugs, including the Bcl-2/Bcl-xL inhibitor ABT-263, salinomycin (SAL), doxorubicin (DOX), paclitaxel (TAX), vincristine (VCR), colchicine (COL) and cisplatin (CIS). ABT-263, SAL and DOX were highly effective towards phyllodes spindle cells when assessed in the model, contributing to ~98% tumor cell death. Furthermore, ABT-263 was highly selective for tumor cells in this system, and exhibited little toxic effect on adjacent normal epithelial cells. Furthermore, consistent with findings in the model, ABT-263 was significantly less toxic towards MCF 10A non-tumorigenic breast epithelial cells compared with SAL and DOX. A conditional reprogramming strategy was subsequently used, involving Rho kinase inhibition, to successfully generate primary phyllodes tumor cells that could be cultured for several passages. The primary cells were sensitive to DOX with an IC of 0.40±0.07 µM in a standard viability assay and the preliminary results were obtained indicating sensitivity to ABT-263 and SAL. The present study demonstrated the feasibility of using explants and primary cells for drug discovery, selectively targeting PTB cells.
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http://dx.doi.org/10.3892/ol.2019.10823DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6781661PMC
November 2019

Flower isoforms promote competitive growth in cancer.

Nature 2019 08 24;572(7768):260-264. Epub 2019 Jul 24.

Champalimaud Centre for the Unknown, Lisbon, Portugal.

In humans, the adaptive immune system uses the exchange of information between cells to detect and eliminate foreign or damaged cells; however, the removal of unwanted cells does not always require an adaptive immune system. For example, cell selection in Drosophila uses a cell selection mechanism based on 'fitness fingerprints', which allow it to delay ageing, prevent developmental malformations and replace old tissues during regeneration. At the molecular level, these fitness fingerprints consist of combinations of Flower membrane proteins. Proteins that indicate reduced fitness are called Flower-Lose, because they are expressed in cells marked to be eliminated. However, the presence of Flower-Lose isoforms at a cell's membrane does not always lead to elimination, because if neighbouring cells have similar levels of Lose proteins, the cell will not be killed. Humans could benefit from the capability to recognize unfit cells, because accumulation of damaged but viable cells during development and ageing causes organ dysfunction and disease. However, in Drosophila this mechanism is hijacked by premalignant cells to gain a competitive growth advantage. This would be undesirable for humans because it might make tumours more aggressive. It is unknown whether a similar mechanism of cell-fitness comparison is present in humans. Here we show that two human Flower isoforms (hFWE1 and hFWE3) behave as Flower-Lose proteins, whereas the other two isoforms (hFWE2 and hFWE4) behave as Flower-Win proteins. The latter give cells a competitive advantage over cells expressing Lose isoforms, but Lose-expressing cells are not eliminated if their neighbours express similar levels of Lose isoforms; these proteins therefore act as fitness fingerprints. Moreover, human cancer cells show increased Win isoform expression and proliferate in the presence of Lose-expressing stroma, which confers a competitive growth advantage on the cancer cells. Inhibition of the expression of Flower proteins reduces tumour growth and metastasis, and induces sensitivity to chemotherapy. Our results show that ancient mechanisms of cell recognition and selection are active in humans and affect oncogenic growth.
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http://dx.doi.org/10.1038/s41586-019-1429-3DOI Listing
August 2019

A rare case of visceral leishmaniasis in an immunocompetent traveler returning to the United States from Europe.

PLoS Negl Trop Dis 2018 10 4;12(10):e0006727. Epub 2018 Oct 4.

Department of Internal Medicine, Section of Hematology and Oncology, Yale University School of Medicine, New Haven, Connecticut, United States of America.

A young, healthy traveler returning to the United States presented with fever, night sweats, splenomegaly, and pancytopenia. Bone marrow biopsy revealed leishmaniasis (Leishmania infantum), likely acquired in southern France. Although many cases of endemic visceral leishmaniasis (VL) have been reported in Europe, this is a rare case of imported VL in a healthy traveler returning from Europe to the US. Despite successful initial treatment with liposomal amphotericin B (LamB), relapse occurred. Treatments for VL in immunocompetent individuals are highly effective, but relapse can occur. There is more extensive experience in endemic areas with treating relapse that may be lacking in North America. This case alerts physicians in the US that immunocompetent adults can acquire VL during brief visits to endemic areas in Europe. It is important that travelers be counseled on preventive measures. Patients should be monitored after treatment for relapse.
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http://dx.doi.org/10.1371/journal.pntd.0006727DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6171829PMC
October 2018

Biological activity of doubly modified salinomycin analogs - Evaluation in vitro and ex vivo.

Eur J Med Chem 2018 Aug 10;156:510-523. Epub 2018 Jul 10.

Centre for Analysis and Synthesis, Department of Chemistry, Lund University, Box 124, 221 00, Lund, Sweden. Electronic address:

The polyether ionophore salinomycin has recently captured much interest due to its potent activity against multi-drug resistant cancer cells and cancer stem cells. Previous studies have shown that either acylation of the C20 position or esterification/amidation of the C1 carboxylate moiety is beneficial in terms of biological properties. In this paper, we present the first analogs combining such modifications. Evaluation of the anti-proliferative activity against a series of cancer cell lines showed that acylation of the C20 hydroxyl group improves the activity of salinomycin C1 amides but not of the corresponding C1 esters. Importantly, the activity of several of the doubly modified analogs surpasses that of commonly used cytostatic drugs cisplatin and doxorubicin in the LoVo/DX multi-drug resistant cell line. All analogs were tested against primary acute lymphoblastic leukemia cells in standard cell viability assays; three were more potent than salinomycin. Further studies revealed that selected analogs induced characteristics of apoptotic cell death and increased expression of p53. Additionally, using an ex vivo model of breast tumor, tumor cell viability significantly decreased after treatment with salinomycin or its double-modified derivative (3a) in a time-dependent manner. The present findings indicate that double-modified salinomycin derivatives constitute promising lead compounds for targeting various types of cancer.
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http://dx.doi.org/10.1016/j.ejmech.2018.07.021DOI Listing
August 2018

Opposing Actions of Developmental Trichloroethylene and High-Fat Diet Coexposure on Markers of Lipogenesis and Inflammation in Autoimmune-Prone Mice.

Toxicol Sci 2018 07;164(1):313-327

Department of Microbiology and Immunology, University of Arkansas for Medical Sciences, Little Rock, Arkansas 72205.

Trichloroethylene (TCE) is a widespread environmental pollutant associated with immunotoxicity and autoimmune disease. Previous studies showed that mice exposed from gestation through early life demonstrated CD4+ T cell alterations and autoimmune hepatitis. Determining the role of one environmental risk factor for any disease is complicated by the presence of other stressors. Based on its known effects, we hypothesized that developmental overnutrition in the form of a moderately high-fat diet (HFD) consisting of 40% kcal fat would exacerbate the immunotoxicity and autoimmune-promoting effects of low-level (<10 μg/kg/day) TCE in autoimmune-prone MRL+/+ mice over either stressor alone. When female offspring were evaluated at 27 weeks of age we found that a continuous exposure beginning at 4 weeks preconception in the dams until 10 weeks of age in offspring that TCE and HFD promoted unique effects that were often antagonistic. For a number of adiposity endpoints, TCE significantly reversed the expected effects of HFD on expression of genes involved in fatty acid synthesis/insulin resistance, as well as mean pathology scores of steatosis. Although none of the animals developed pathological signs of autoimmune hepatitis, the mice generated unique patterns of antiliver antibodies detected by western blotting attributable to TCE exposure. A majority of cytokines in liver, gut, and splenic CD4+ T cells were significantly altered by TCE, but not HFD. Levels of bacterial populations in the intestinal ileum were also altered by TCE exposure rather than HFD. Thus, in contrast to our expectations this coexposure did not promote synergistic effects.
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http://dx.doi.org/10.1093/toxsci/kfy091DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6016708PMC
July 2018

Myeloid transformation of plasma cell myeloma: molecular evidence of clonal evolution revealed by next generation sequencing.

Diagn Pathol 2018 Feb 20;13(1):15. Epub 2018 Feb 20.

Department of Pathology, University of Arkansas for Medical Sciences, Little Rock, AR, 72205-7199, USA.

Background: Plasma cell myeloma (PCM) is a neoplasm of terminally differentiated B lymphocytes with molecular heterogeneity. Although therapy-related myeloid neoplasms are common in plasma cell myeloma patients after chemotherapy, transdifferentiation of plasma cell myeloma into myeloid neoplasms has not been reported in literature. Here we report a very rare case of myeloid neoplasm transformed from plasma cell myeloma.

Case Presentation: A 60-year-old man with a history of plasma cell myeloma with IGH-MAF gene rearrangement and RAS/RAF mutations developed multiple soft tissue lesions one year following melphalan-based chemotherapy and autologous stem cell transplant. Morphological and immunohistochemical characterization of the extramedullary disease demonstrated that the tumor cells were derived from the monocyte-macrophage lineage. Next generation sequencing (NGS) studies detected similar clonal aberrations in the diagnostic plasma cell population and post-therapy neoplastic cells, including IGH-MAF rearrangement, multiple genetic mutations in RAS signaling pathway proteins, and loss of tumor suppressor genes. Molecular genetic analysis also revealed unique genomic alterations in the transformed tumor cells, including gain of NF1 and loss of TRAF3.

Conclusion: To our knowledge, this is the first case of myeloid sarcoma transdifferentiated from plasma cell neoplasm. Our findings in this unique case suggest clonal evolution of plasma cell myeloma to myeloma neoplasm and the potential roles of abnormal RAS/RAF signaling pathway in lineage switch or transdifferentiation.
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http://dx.doi.org/10.1186/s13000-018-0692-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5819706PMC
February 2018

CD3-positive plasmablastic B-cell neoplasms: a diagnostic pitfall.

Mod Pathol 2018 05 12;31(5):718-731. Epub 2018 Jan 12.

Department of Pathology and Microbiology, University of Nebraska Medical Center, Omaha, NE, USA.

Rare B-cell neoplasms with plasmablastic differentiation may aberrantly express CD3 by immunohistochemical staining, which places a great challenge for diagnosis. We here studied 17 cases of CD3+ plasmablastic B-cell neoplasms, including 12 plasmablastic lymphomas and 5 plasmablastic plasma cell myelomas. All 17 cases occurred in the extranodal sites with a male predominance (13/17). Four cases were initially misinterpreted by outside institutions, among which three were diagnosed as 'peripheral T-cell lymphoma, not otherwise specified' and one was classified as 'poorly differentiated neuroendocrine carcinoma'. The plasmablastic cells were present in all 17 cases diffusely or in a subset of tumor cells. CD3 expression was mostly diffuse (12/17) and moderate to strong (11/16) with a cytoplasmic staining pattern (14/16). Other T-cell markers were nearly absent, including CD2 (0/10), CD4 (1/13), CD5 (0/14), CD7 (0/11), and CD8 (0/13). CD138 was positive in all 17 cases and CD79a was variably positive in 8 of 14 cases. Only one case had immunoreactivity to CD20 (1/17) and PAX5 (1/12). CD56 expression and EBV infection were detected in 8/15 and 6/17, respectively. No HHV8 infection was noted in all 11 cases tested. Most cases (11/13) revealed either kappa or lambda light chain restriction. Of the nine cases studied, six had clonal IGH rearrangements but no clonal TRG rearrangements. Our study further emphasizes that the accurate classification of CD3+ plasmablastic neoplasms requires thorough morphologic examination, incorporation of more B-cell and T-cell markers in addition to CD3 and CD20, frequent addition of CD138 staining, and utilization of necessary molecular and genetic studies.
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http://dx.doi.org/10.1038/modpathol.2017.177DOI Listing
May 2018

Diagnostic Utility of Interleukin-6 Expression by Immunohistochemistry in Differentiating Castleman Disease Subtypes and Reactive Lymphadenopathies.

Ann Clin Lab Sci 2016 Sep;46(5):474-9

Department of Pathology, University of Arkansas for Medical Sciences, Little Rock, AR, USA.

The objective of the study was to evaluate the expression pattern of interleukin-6 (IL-6) to determine its utility in differentiating Castleman Disease subtypes and reactive lymphadenopathies. Paraffin-embedded tissue blocks from 20 cases referred for assessment of Castleman Disease (CD) and 4 cases of reactive hyperplasia were selected for immunohistochemical staining with an IL-6 antibody. Six pathologists evaluated the hematoxylin and eosin stained tissue sections and IL-6 expression pattern. Of 20 CD referral cases, the pathologic diagnosis was CD in 14 cases and included 6 hyaline-vascular (HV-CD), 6 plasma cell (PC-CD) and 2 "mixed type"-CD cases. The remaining 6 referral cases showed morphologic features consistent with reactive lymphadenopathy. Patients with non-CD, reactive lymphadenopathies had clinical and/or laboratory features of systemic lupus erythematosus, Hashimoto's disease, viral infection or chronic cellulitis. The pattern of IL-6 expression differed between CD subtypes and non-CD cases. In PC-CD, IL-6 expression was detected in plasma cells and vascular endothelial cells; whereas IL-6 immunoreactivity was detected primarily in vascular endothelial cells in HV-CD. Interfollicular plasma cells were prominent in PC-CD and reactive lymphadenopathies; however, IL-6 expression was significantly increased in PC-CD compared to reactive lymph nodes. Together with morphologic features, the expression pattern of IL-6 detected by immunohistochemistry is helpful to distinguish CD subtypes and reactive mimics.
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September 2016

Small-molecule inhibitors targeting INK4 protein p18(INK4C) enhance ex vivo expansion of haematopoietic stem cells.

Nat Commun 2015 Feb 18;6:6328. Epub 2015 Feb 18.

1] Department of Pharmaceutical Sciences, Computational Chemical Genomics Screening Center, School of Pharmacy, NIH National Center of Excellence for Drug Abuse Research, Drug Discovery Institute, Pittsburgh, Pennsylvania 15260, USA [2] Department of Computational and System Biology, University of Pittsburgh, Pittsburgh, Pennsylvania 15260, USA.

Among cyclin-dependent kinase inhibitors that control the G1 phase in cell cycle, only p18 and p27 can negatively regulate haematopoietic stem cell (HSC) self-renewal. In this manuscript, we demonstrate that p18 protein is a more potent inhibitor of HSC self-renewal than p27 in mouse models and its deficiency promoted HSC expansion in long-term culture. Single-cell analysis indicated that deleting p18 gene favoured self-renewing division of HSC in vitro. Based on the structure of p18 protein and in-silico screening, we further identified novel smallmolecule inhibitors that can specifically block the activity of p18 protein. Our selected lead compounds were able to expand functional HSCs in a short-term culture. Thus, these putative small-molecule inhibitors for p18 protein are valuable for further dissecting the signalling pathways of stem cell self-renewal and may help develop more effective chemical agents for therapeutic expansion of HSC.
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http://dx.doi.org/10.1038/ncomms7328DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4508125PMC
February 2015

The p53-PUMA axis suppresses iPSC generation.

Nat Commun 2013 ;4:2174

State Key Laboratory of Experimental Hematology, Institute of Hematology and Blood Diseases Hospital, Center for Stem Cell Medicine, Chinese Academy of Medical Sciences and Peking Union Medical College, Nanjing Road No. 288, Tianjin 300020, China.

Mechanisms underlying the reprogramming process of induced pluripotent stem cells remain poorly defined. Like tumorigenesis, generation of induced pluripotent stem cells was shown to be suppressed by the Trp53 (p53) pathway, at least in part via p21Cdkn1a (p21)-mediated cell cycle arrest. Here we examine the role of PUMA, a pro-apoptotic mediator of p53, during somatic reprogramming in comparison to p21 in the p53 pathway. Using mouse strains deficient in these molecules, we demonstrate that PUMA is an independent mediator of the negative effect of p53 on induced pluripotent stem cell induction. PUMA deficiency leads to a better survival rate associated with reduced DNA damage and fewer chromosomal aberrations in induced pluripotent stem cells, whereas loss of p21 or p53 results in an opposite outcome. Given these new findings, PUMA may serve as a distinct and more desirable target in the p53 pathway for induced pluripotent stem cell generation, thereby having important implications for potential therapeutic applications of induced pluripotent stem cells.
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http://dx.doi.org/10.1038/ncomms3174DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4394110PMC
January 2014

An acute negative bystander effect of γ-irradiated recipients on transplanted hematopoietic stem cells.

Blood 2012 Apr 28;119(15):3629-37. Epub 2012 Feb 28.

Department of Radiation Oncology, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA.

Ultimate success of hematopoietic stem cell transplantation (HSCT) depends not only on donor HSCs themselves but also on the host environment. Total body irradiation is a component in various host conditioning regimens for HSCT. It is known that ionizing radiation exerts "bystander effects" on nontargeted cells and that HSCs transplanted into irradiated recipients undergo proliferative exhaustion. However, whether irradiated recipients pose a proliferation-independent bystander effect on transplanted HSCs is unclear. In this study, we found that irradiated mouse recipients significantly impaired the long-term repopulating ability of transplanted mouse HSCs shortly (∼ 17 hours) after exposure to irradiated hosts and before the cells began to divide. There was an increase of acute cell death associated with accelerated proliferation of the bystander hematopoietic cells. This effect was marked by dramatic down-regulation of c-Kit, apparently because of elevated reactive oxygen species. Administration of an antioxidant chemical, N-acetylcysteine, or ectopically overexpressing a reactive oxygen species scavenging enzyme, catalase, improved the function of transplanted HSCs in irradiated hosts. Together, this study provides evidence for an acute negative, yet proliferation-independent, bystander effect of irradiated recipients on transplanted HSCs, thereby having implications for HSCT in both experimental and clinical scenarios in which total body irradiation is involved.
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http://dx.doi.org/10.1182/blood-2011-08-373621DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3325047PMC
April 2012

Role of macrophages in mobilization of hematopoietic progenitor cells from bone marrow after hemorrhagic shock.

Shock 2012 May;37(5):518-23

Department of Surgery, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, USA.

The release of hematopoietic progenitor cells (HPCs) from bone marrow (BM) is under tight homeostatic control. Under stress conditions, HPCs migrate from BM and egress into circulation to participate in immune response, wound repair, or tissue regeneration. Hemorrhagic shock with resuscitation (HS/R), resulting from severe trauma and major surgery, promotes HPC mobilization from BM, which, in turn, affects post-HS immune responses. In this study, we investigated the mechanism of HS/R regulation of HPC mobilization from BM. Using a mouse HS/R model, we demonstrate that the endogenous alarmin molecule high-mobility group box 1 mediates HS/R-induced granulocyte colony-stimulating factor secretion from macrophages (Mϕ in a RAGE [receptor for advanced glycation end products] signaling-dependent manner. Secreted granulocyte colony-stimulating factor, in turn, induces HPC egress from BM. We also show that activation of β-adrenergic receptors on Mϕ by catecholamine mediates the HS/R-induced release of high-mobility group box 1. These data indicate that HS/R, a global ischemia-reperfusion stimulus, regulates HPC mobilization through a series of interacting pathways that include neuroendocrine and innate immune systems, in which Mϕ play a central role.
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http://dx.doi.org/10.1097/SHK.0b013e318249b81dDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3328610PMC
May 2012

Deletion of Puma protects hematopoietic stem cells and confers long-term survival in response to high-dose gamma-irradiation.

Blood 2010 Apr 22;115(17):3472-80. Epub 2010 Feb 22.

Department of Radiation Oncology, University of Pittsburgh School of Medicine, PA, USA.

Molecular paradigms underlying the death of hematopoietic stem cells (HSCs) induced by ionizing radiation are poorly defined. We have examined the role of Puma (p53 up-regulated mediator of apoptosis) in apoptosis of HSCs after radiation injury. In the absence of Puma, HSCs were highly resistant to gamma-radiation in a cell autonomous manner. As a result, Puma-null mice or the wild-type mice reconstituted with Puma-null bone marrow cells were strikingly able to survive for a long term after high-dose gamma-radiation that normally would pose 100% lethality on wild-type animals. Interestingly, there was no increase of malignancy in the exposed animals. Such profound beneficial effects of Puma deficiency were likely associated with better maintained quiescence and more efficient DNA repair in the stem cells. This study demonstrates that Puma is a unique mediator in radiation-induced death of HSCs. Puma may be a potential target for developing an effective treatment aimed to protect HSCs from lethal radiation.
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http://dx.doi.org/10.1182/blood-2009-10-248278DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2867261PMC
April 2010

Hemorrhagic shock augments lung endothelial cell activation: role of temporal alterations of TLR4 and TLR2.

Am J Physiol Regul Integr Comp Physiol 2009 Dec 14;297(6):R1670-80. Epub 2009 Oct 14.

Department of Surgery, School of Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania, USA.

Hemorrhagic shock (HS) due to major trauma predisposes the host to the development of acute lung inflammation and injury. The lung vascular endothelium is an active organ that plays a central role in the development of acute lung injury through generating reactive oxygen species and synthesizing and releasing of a number of inflammatory mediators, including leukocyte adhesion molecules that regulate neutrophils emigration. Previous study from our laboratory has demonstrated that in a setting of sepsis, toll-like receptor-4 (TLR4) signaling can induce TLR2 expression in endothelial cells (ECs), thereby increasing the cells' response to TLR2 ligands. The present study tested the hypothesis that TLR4 activation by HS and the resultant increased TLR2 surface expression in ECs might contribute to the mechanism underlying HS-augmented activation of lung ECs. The results show that high-mobility group box 1 (HMGB1) through TLR4 signaling mediates HS-induced surface expression of TLR2 in the lung and mouse lung vascular endothelial cells (MLVECs). Furthermore, the results demonstrate that HMGB1 induces activation of NAD(P)H oxidase and expression of ICAM-1 in the lung, and MLVECs sequentially depend on TLR4 in the early phase and on TLR2 in the late phase following HS. Finally, the data indicate an important role of the increased TLR2 surface expression in enhancing the activation of MLVECs and augmenting pulmonary neutrophil infiltration in response to TLR2 agonist peptidoglycan. Thus, induction of TLR2 surface expression in lung ECs, induced by HS and mediated by HMGB1/TLR4 signaling, is an important mechanism responsible for endothelial cell-mediated inflammation and organ injury following trauma and hemorrhage.
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http://dx.doi.org/10.1152/ajpregu.00445.2009DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2803625PMC
December 2009

Preactivation of NKT cells with alpha-GalCer protects against hepatic ischemia-reperfusion injury in mouse by a mechanism involving IL-13 and adenosine A2A receptor.

Am J Physiol Gastrointest Liver Physiol 2009 Aug 25;297(2):G249-58. Epub 2009 Jun 25.

Department of Surgery, University of Pittsburgh, Pittsburgh, Pennsylvania 15213, USA.

Hepatic preconditioning has emerged as a promising strategy of activating natural pathways to augment tolerance to liver ischemia-reperfusion (IR) injury. Liver-resident natural killer T (NKT) cells play an important role in modulating the local immune and inflammatory responses. This work was aimed to investigate whether preactivation of NKT cells could provide a beneficial "preconditioning" effect to ameliorate the subsequent hepatic IR injury. To selectively activate NKT cells, C57BL/6 mice were treated intraperitoneally with the glycolipid antigen alpha-galactosylceramide (alpha-GalCer) 1 h prior to hepatic ischemia. Significantly reduced liver IR injury was observed in mice pretreated with alpha- GalCer, and this protective effect was specifically abrogated by a CD1d blocking antibody. Serum TNF-alpha, IFN-gamma, and IL-13 levels were markedly increased shortly after alpha-GalCer injection. Pretreatment with a neutralizing antibody against TNF-alpha or IFN-gamma did not influence the protective effect of alpha-GalCer preconditioning, whereas preadministration of an IL-13 neutralizing antibody completely abolished the effect. Treatment with alpha-GalCer also led to an increased expression of adenosine A2A receptor (A2AR) in the liver, and blockade of A2AR by SH58261 diminished alpha-GalCer pretreatment-mediated attenuation of liver IR injury. In contrast, administration of the selective A2AR agonist CGS21680 reversed the counteracting effect of the IL-13 neutralizing antibody on alpha-GalCer preconditioning. Additionally, alpha-GalCer pretreatment was associated with a decreased neutrophil accumulation in the ischemic liver. These findings provide the first evidence that hepatic preconditioning by preactivation of NKT cells with alpha-GalCer protects the liver from IR injury via an IL-13 and adenosine A2AR-dependent mechanism.
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http://dx.doi.org/10.1152/ajpgi.00041.2009DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2724078PMC
August 2009

Interacting neuroendocrine and innate and acquired immune pathways regulate neutrophil mobilization from bone marrow following hemorrhagic shock.

J Immunol 2009 Jan;182(1):572-80

Department of Surgery, School of Medicine, University of Pittsburgh, Pittsburgh, PA 15213, USA.

Polymorphonuclear neutrophils (PMN) are critical innate immune effector cells that either protect the host or exacerbate organ dysfunction by migrating to injured or inflamed tissues. Resuscitated hemorrhagic shock following major trauma promotes the development of organ inflammation by priming PMN migration and activation in response to a second, often trivial, stimulus (a so-called "two hit" phenomenon). PMN mobilization from bone marrow supports a sustained, hemorrhagic shock/resuscitation (HS/R)-primed migration of PMN. We addressed the role and mechanism of HS/R in regulating PMN egress from bone marrow. We demonstrate that HS/R through the alarmin HMGB1 induces IL-23 secretion from macrophages in an autocrine and TLR4 signaling-dependent manner. In turn IL-23, through an IL-17 G-CSF-mediated mechanism, induces PMN egress from bone marrow. We also show that beta-adrenergic receptor activation by catecholamine of macrophages mediates the HS/R-induced release of HMGB1. These data indicate that HS/R, a global ischemia/reperfusion stimulus, regulates PMN mobilization through a series of interacting pathways that include neuroendocrine and innate and acquired immune systems. Blocking this novel signaling axis may present a novel therapeutic target for posttrauma inflammation.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2610356PMC
http://dx.doi.org/10.4049/jimmunol.182.1.572DOI Listing
January 2009

Changes in FADD levels, distribution, and phosphorylation in TNFalpha-induced apoptosis in hepatocytes is caspase-3, caspase-8 and BID dependent.

Apoptosis 2008 Aug;13(8):983-92

Department of Surgery, University of Pittsburgh, School of Medicine, Pittsburgh, PA 15213, USA.

FADD/MORT1 (The adaptor protein of Fas Associate Death Domain/Mediator of Receptor Induced Toxicity) is essential for signal transduction of death receptor signaling. We have previously shown that FADD is significantly up-regulated in TNFalpha/ActD induced apoptosis. Over-expression of FADD also induces death of lung cancer cells and primary hepatocytes. We hypothesize that the increase in detectable FADD levels require the proximal steps in apoptotic signaling and speculated that FADD would be redistributed in cells destined to undergo apoptosis. We show that monomeric non-phosphorylated FADD is up-regulated in hepatocytes treated with TNFalpha/ActD and that it accumulates in the cytoplasm. Nuclear phosphorylated FADD decreases with TNFalpha/ActD treatment. Dimeric FADD in the cytoplasm remains constant with TNFalpha/ActD. The change in FADD levels and distribution was dependent on caspase-3, caspase-8 activity and the presence of BID. Thus, changes in FADD levels and distribution are downstream of caspase activation and mitochondria changes that are initiated by the formation of the DISC complex. Changes in FADD levels and distribution may represent a novel feed-forward mechanism to propagate apoptosis signaling in hepatocytes.
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http://dx.doi.org/10.1007/s10495-008-0228-3DOI Listing
August 2008

Beta-catenin deletion in hepatoblasts disrupts hepatic morphogenesis and survival during mouse development.

Hepatology 2008 May;47(5):1667-79

Department of Pathology, University of Pennsylvania, School of Medicine, Philadelphia, PA, USA.

Unlabelled: Beta-catenin, the central component of the canonical Wnt pathway, plays important roles in the processes of liver regeneration, growth, and cancer. Previously, we identified temporal expression of beta-catenin during liver development. Here, we characterize the hepatic phenotype, resulting from the successful deletion of beta-catenin in the developing hepatoblasts utilizing Foxa3-cyclization recombination and floxed-beta-catenin (exons 2 through 6) transgenic mice. Beta-catenin loss in developing livers resulted in significantly underdeveloped livers after embryonic day 12 (E12) with lethality occurring at around E17 stages. Histology revealed an overall deficient hepatocyte compartment due to (1) increased cell death due to oxidative stress and apoptosis, and (2) diminished expansion secondary to decreased cyclin-D1 and impaired proliferation. Also, the remnant hepatocytes demonstrated an immature phenotype as indicated by high nuclear to cytoplasmic ratio, poor cell polarity, absent glycogen, and decreased expression of key liver-enriched transcription factors: CCAAT-enhancer binding protein-alpha and hepatocyte nuclear factor-4alpha. A paucity of primitive bile ducts was also observed. While the stem cell assays demonstrated no intrinsic defect in hematopoiesis, distorted hepatic architecture and deficient hepatocyte compartments resulted in defective endothelial cell organization leading to overall fetal pallor.

Conclusion: Beta-catenin regulates multiple, critical events during the process of hepatic morphogenesis, including hepatoblast maturation, expansion, and survival, making it indispensable to survival.
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http://dx.doi.org/10.1002/hep.22225DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4449338PMC
May 2008

Long-term engraftment of p18(INK4C)-deficient hematopoietic stem cells is enhanced in the sublethally-irradiated recipients.

Sci China C Life Sci 2006 Aug;49(4):390-4

Department of Hematology, Changhai Hospital, Second Military Medical University, Shanghai 200433, China.

Non-myeloablative regimens for host conditioning have been widely used in clinical hematopoietic stem cell transplantation due to their reduced toxicity on the recipients. But a milder conditioning regimen may require a higher engrafting ability of donor stem cells in competing with endogenous stem cells. Thus, new strategies for enhancing the competitiveness of donor stem cells in non-myeloablative recipients would have important implications for current clinical stem cell transplantation. It is known that the absence of p18(INK4C) (p18) gene can enhance the self-renewal potential of hematopoietic stem cells (HSCs). We applied the approach of competitive bone marrow transplantation to evaluate the impact of p18 gene deletion on long-term engraftment of HSCs in sublethally irradiated hosts. We found that p18(-/-) HSCs had a significant advantage over wild-type HSCs during long-term engraftment in the mouse recipients that received a sub-lethal irradiation (5-Gy). The engraftment efficiency of p18(-/-) HSCs in the sub-lethally irradiated recipients was similar to that in the lethally irradiated (10-Gy) recipients. Our current study demonstrates that enhanced engraftment of donor HSCs in the absence of p18 does not strictly depend on the dose of irradiation used for host conditioning. Therefore, p18 might serve as a potential drug target for increasing the efficacy of stem cell transplant in the patients that are preconditioned with either a myeloablative or non-myeloablative regimen.
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http://dx.doi.org/10.1007/s11427-006-0260-xDOI Listing
August 2006

Hematopoietic stem cells are not the direct target of spontaneous leukemic transformation in p18(INK4C)-null reconstituted mice.

Cancer Res 2006 Jan;66(1):343-51

Department of Radiation Oncology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, USA.

Cell cycle inhibitors are important regulators in normal tissue regeneration and disruption of the regulators are involved in cancer development. Our recent study showed that the absence of the CDK inhibitor p18(INK4C) (p18) enhances self-renewal of normal hematopoietic stem cell (HSC) in vivo, whereas previous studies by others showed an increased incidence of leukemogenesis in older p18-null mice. Here, we have examined potential leukemogenesis during experimentally induced regeneration of HSC in the absence of p18 in order to gauge the relation between these two processes. Reconstituted mice with p18-deficient HSCs under the condition of repetitive proliferative stress (serial transplantation) were followed for >3 years. T cell leukemia from the p18-/- origin was recapitulated 24 months after secondary transplantation. However, no myeloid leukemia was found in the recipients. The T cell leukemia-initiating cells (mainly in a CD3(lo) cell subset) did not share the same immunophenotype with normal HSCs and, in fact, the function of HSCs was significantly compromised with decreased abundance in the leukemic mice. Furthermore, we found that the p15 or p16 gene promoters were frequently methylated in the leukemic cells but not in HSCs. Our present study argues against the possibility of overgrowth of p18-null HSCs leading to a leukemic phenotype. The data also support the notion that p18 has an independent role in T cell maintenance such that CD3+ CD8+ cells, unlike HSCs, are more accessible to leukemogenic transformation after the loss of p18.
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http://dx.doi.org/10.1158/0008-5472.CAN-05-2945DOI Listing
January 2006

Hematopoietic stem cell exhaustion impacted by p18 INK4C and p21 Cip1/Waf1 in opposite manners.

Blood 2006 Feb 18;107(3):1200-6. Epub 2005 Oct 18.

Department of Radiation Oncology, University of Pittsburgh School of Medicine, University of Pittsburgh Cancer Institute, PA, USA.

Transplantation-associated stress can compromise the hematopoietic potential of hematopoietic stem cells (HSCs). As a consequence, HSCs may undergo "exhaustion" in serial transplant recipients, for which the cellular and molecular bases are not well understood. Hematopoietic exhaustion appears to be accelerated in the absence of p21(Cip1/Waf1) (p21), a cyclin-dependent kinase inhibitor (CKI) in irradiated hosts. Our recent study demonstrated that unlike loss of p21, deletion of p18(INK4C) (p18), a distinct CKI, results in improved long-term engraftment, largely because of increased self-renewing divisions of HSCs in vivo. We show here that HSCs deficient in p18 sustained their competitiveness to wild-type HSCs from unmanipulated young mice, and retained multilineage differentiation potential after multiple rounds of serial bone marrow transfer over a period of more than 3 years. Further, p18 absence significantly decelerated hematopoietic exhaustion caused by p21 deficiency. Such an effect was shown to occur at the stem cell level, likely by a counteracting mechanism against the cellular senescence outcome. Our current study provides new insights into the distinct impacts of these cell-cycle regulators on HSC exhaustion and possibly HSC aging as well under proliferative stress, thereby offering potential pharmacologic targets for sustaining the durability of stressed HSCs in transplantation or elderly patients.
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http://dx.doi.org/10.1182/blood-2005-02-0685DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1895913PMC
February 2006

In vivo self-renewing divisions of haematopoietic stem cells are increased in the absence of the early G1-phase inhibitor, p18INK4C.

Nat Cell Biol 2004 May;6(5):436-42

University of Pittsburgh Cancer Institute and Department of Radiation Oncology, University of Pittsburgh School of Medicine, Pittsburgh PA 15213, USA.

Self-renewal of stem cells is critical for tissue repair and maintenance of organ integrity in most mammalian systems. The relative asymmetry between self-renewal and differentiation in balance with apoptosis determines the size and durability of a stem-cell pool. Regulation of the cell cycle is one of the fundamental mechanisms underlying determination of cell fate. Absence of p21(Cip1/Waf1), a late G1-phase cyclin-dependent kinase inhibitor (CKI), has previously been shown to enable cell-cycle entry of haematopoietic stem cells, but leads to premature exhaustion of the stem cells under conditions of stress. We show here that deletion of an early G1-phase CKI, p18(INK4C), results in strikingly improved long-term engraftment, largely by increasing self-renewing divisions of the primitive cells in murine transplant models. Therefore, different CKIs have highly distinct effects on the kinetics of stem cells, possibly because of their active position in the cell cycle, and p18(INK4C) appears to be a strong inhibitor limiting the potential of stem-cell self-renewal in vivo.
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http://dx.doi.org/10.1038/ncb1126DOI Listing
May 2004

Protein ISGylation modulates the JAK-STAT signaling pathway.

Genes Dev 2003 Feb;17(4):455-60

Department of Molecular and Experimental Medicine, The Scripps Research Institute, La Jolla, California 92037, USA.

ISG15 is one of the most strongly induced genes upon viral infection, type I interferon (IFN) stimulation, and lipopolysaccharide (LPS) stimulation. Here we report that mice lacking UBP43, a protease that removes ISG15 from ISGylated proteins, are hypersensitive to type I IFN. Most importantly, in UBP43-deficient cells, IFN-beta induces a prolonged Stat1 tyrosine phosphorylation, DNA binding, and IFN-mediated gene activation. Furthermore, restoration of ISG15 conjugation in protein ISGylation-defective K562 cells increases IFN-stimulated promoter activity. These findings identify UBP43 as a novel negative regulator of IFN signaling and suggest the involvement of protein ISGylation in the regulation of the JAK-STAT pathway.
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http://dx.doi.org/10.1101/gad.1056303DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC195994PMC
February 2003
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