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Browsing by Author "Hangoc, Giao"
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Item Activation of OCT4 enhances ex vivo expansion of human cord blood hematopoietic stem and progenitor cells by regulating HOXB4 expression(SpringerNature, 2016-01) Huang, Xinxin; Lee, Man-Ryul; Cooper, Scott; Hangoc, Giao; Hong, Ki-Sung; Chung, Hyung-Min; Broxmeyer, Hal E.; Department of Microbiology & Immunology, IU School of MedicineAlthough hematopoietic stem cells (HSC) are the best characterized and the most clinically used adult stem cells, efforts are still needed to understand how to best ex vivo expand these cells. Here we present our unexpected finding that OCT4 is involved in the enhancement of cytokine-induced expansion capabilities of human cord blood (CB) HSC. Activation of OCT4 by Oct4-activating compound 1 (OAC1) in CB CD34(+) cells enhanced ex vivo expansion of HSC, as determined by a rigorously defined set of markers for human HSC, and in vivo short-term and long-term repopulating ability in NSG mice. Limiting dilution analysis revealed that OAC1 treatment resulted in 3.5-fold increase in the number of SCID repopulating cells (SRCs) compared with that in day 0 uncultured CD34(+) cells and 6.3-fold increase compared with that in cells treated with control vehicle. Hematopoietic progenitor cells, as assessed by in vitro colony formation, were also enhanced. Furthermore, we showed that OAC1 treatment led to OCT4-mediated upregulation of HOXB4. Consistently, siRNA-mediated knockdown of HOXB4 expression suppressed effects of OAC1 on ex vivo expansion of HSC. Our study has identified the OCT4-HOXB4 axis in ex vivo expansion of human CB HSC.Item Drugging the “Undruggable” DNA-binding Domain of STAT3 for Inhibition of Cancer Cell Migration and Invasion(Office of the Vice Chancellor for Research, 2013-04-05) Huang, Wei; Liu, Jing-Yuan; Dong, Zi-Zheng; Wang, Fang; He, Yan-Tao; Hangoc, Giao; Fu, Xin-Yuan; Broxmeyer, Hal; Zhang, Zhong-Yin; Zhang, Jian-TingSignal transducer and activator of transcription 3 (STAT3) is constitutively activated in malignant tumors, and its activation is associated with high histological grade and advanced cancer stage. STAT3 has been shown to play important roles in multiple aspects of cancer aggressiveness including migration, invasion, survival, self-renewal, angiogenesis, and tumor cell immune evasion by regulating the expression of multiple downstream target genes. Thus, inhibiting STAT3 promises an attracting strategy for treatment of advanced tumors with metastatic potential. Previously, we identified a STAT3 inhibitor, inS3-54, by targeting the “undruggable” DNA-binding site of STAT3 using an improved in-silico screening approach. To further develop this inhibitor, we identified 79 analogues of inS3-54 for the structure-activity relationship analysis. Further study of five effective analogues shows that four analogues (#1, 18, 26, and 69) inhibit STAT3-dependent colony formation of hematopoietic progenitor cells, indicating a higher selectivity for STAT3 than their parental compound, inS3-54 and another analogue #74. These compounds also (1) inhibit STAT3-specific DNA binding activity; (2) suppress proliferation of cancer cells that have constitutively activated STAT3; and (3) inhibit migration and invasion of cancer cells. In addition, analogue #26-conjugated Sepharose beads could also pull down STAT3, revealing a possible direct binding between STAT3 and the inhibitor. Taken together, we conclude that it is possible to inhibit STAT3 by targeting its DNA-binding domain for discovery of anticancer therapeutics and for treatment of metastatic cancers.Item Enhancing Hematopoietic Stem Cell Transplantation Efficacy by Mitigating Oxygen Shock(Elsevier, 2015-06-18) Mantel, Charlie R.; O'Leary, Heather A.; Chitteti, Brahmananda R.; Huang, XinXin; Cooper, Scott; Hangoc, Giao; Brustovetsky, Nickolay; Srour, Edward F.; Lee, Man-Ryul; Messina-Graham, Steve; Haas, David M.; Falah, Nadia; Kapur, Reuben; Pelus, Louis M.; Bardeesy, Nabeel; Fitamant, Julien; Ivan, Mircea; Kim, Kye-Seong; Broxmeyer, Hal E.; Department of Microbiology & Immunology, IU School of MedicineHematopoietic stem cells (HSCs) reside in hypoxic niches within bone marrow and cord blood. Yet, essentially all HSC studies have been performed with cells isolated and processed in non-physiologic ambient air. By collecting and manipulating bone marrow and cord blood in native conditions of hypoxia, we demonstrate that brief exposure to ambient oxygen decreases recovery of long-term repopulating HSCs and increases progenitor cells, a phenomenon we term extraphysiologic oxygen shock/stress (EPHOSS). Thus, true numbers of HSCs in the bone marrow and cord blood are routinely underestimated. We linked ROS production and induction of the mitochondrial permeability transition pore (MPTP) via cyclophilin D and p53 as mechanisms of EPHOSS. The MPTP inhibitor cyclosporin A protects mouse bone marrow and human cord blood HSCs from EPHOSS during collection in air, resulting in increased recovery of transplantable HSCs. Mitigating EPHOSS during cell collection and processing by pharmacological means may be clinically advantageous for transplantation.Item Mad2 Haploinsufficiency Protects Hematopoietic Progenitor Cells Subjected to Cell Cycle Stress In Vivo and to Inhibition of Redox Function of Ape1/Ref-1 In Vitro(2011-04) Rohrabaugh, Sara L.; Hangoc, Giao; Kelley, Mark R.; Broxmeyer, Hal E.Objective Cell-cycle checkpoints guarantee movement through the cell cycle. Mitotic arrest deficiency 2 (Mad2), a mitotic checkpoint protein, appears crucial for generating the wait anaphase signal to prevent onset of anaphase. We evaluated effects of Mad2 haploinsufficiency on hematopoietic stem (HSC) and progenitor (HPC) function in response to stress. Materials and Methods We studied effects of Mad2+/− on in vivo recovery of bone marrow HPC from cytotoxic effects and also effects of cytostatic agents on HPC growth in vitro using Mad2+/− mice. Results Mad2+/− HPCs were protected from cytotoxic effects in vivo of a cell-cycle−specific agent, Ara-C, events consistent with Mad2+/− HPCs being in a slow or noncycling state, but not from recovery of functional HPC after treatment with non-cycle−specific cyclophosphamide or sublethal irradiation. There were no differences in phenotyped HSCs in Mad2+/− & Mad2+/+ mice, information confirmed by no changes in short- or long-term repopulating HSC assay. To better understand Mad2+/− HPC function, E3330, a cytostatic agent, was used to assess redox function of Ape1/Ref-1; colony growth was examined under 5% and 20% O2 tension. Mad2+/− HPCs were less responsive to E3330 than Mad2+/+ HPCs, and E3330 was more effective under lowered O2 tension. Mad2+/− HPCs were not enhanced at lowered oxygen, as were Mad2+/+ HPCs. Conclusions Our studies have unexpectedly found that Mad2 haploinsufficiency is protective in the presence of a cycle-specific DNA synthesis agent in vivo, and Ape1/Ref-1 inhibitor in vitro.Item Mild Heat Treatment Primes Human CD34(+) Cord Blood Cells for Migration Toward SDF-1α and Enhances Engraftment in an NSG Mouse Model(Wiley Blackwell (John Wiley & Sons), 2015-06) Capitano, Maegan L.; Hangoc, Giao; Cooper, Scott; Broxmeyer, Hal E.; Department of Microbiology and Immunology, IU School of MedicineSimple efforts are needed to enhance cord blood (CB) transplantation. We hypothesized that short-term exposure of CD34(+) CB cells to 39.5°C would enhance their response to stromal-derived factor-1 (SDF-1), by increasing lipid raft aggregation and CXCR4 expression, thus leading to enhanced engraftment. Mild hyperthermia (39.5°C) significantly increased the percent of CD34(+) CB that migrated toward SDF-1. This was associated with increased expression of CXCR4 on the cells. Mechanistically, mild heating increased the percent of CD34(+) cells with aggregated lipid rafts and enhanced colocalization of CXCR4 within lipid raft domains. Using methyl-β-cyclodextrin (MβCD), an agent that blocks lipid raft aggregation, it was determined that this enhancement in chemotaxis was dependent upon lipid raft aggregation. Colocalization of Rac1, a GTPase crucial for cell migration and adhesion, with CXCR4 to the lipid raft was essential for the effects of heat on chemotaxis, as determined with an inhibitor of Rac1 activation, NSC23766. Application-wise, mild heat treatment significantly increased the percent chimerism as well as homing and engraftment of CD34(+) CB cells in sublethally irradiated non-obese diabetic severe combined immunodeficiency IL-2 receptor gamma chain d (NSG) mice. Mild heating may be a simple and inexpensive means to enhance engraftment following CB transplantation in patients.Item Modulation of Hematopoietic Chemokine Effects In Vitro and In Vivo by DPP-4/CD26(Mary Ann Liebert, 2016-04-15) Broxmeyer, Hal E.; Capitano, Maegan; Campbell, Timothy B.; Hangoc, Giao; Cooper, Scott; Department of Microbiology and Immunology, School of MedicineDipeptidyl peptidase 4 (DPP4)/CD26 truncates certain proteins, and this posttranslational modification can influence their activity. Truncated (T) colony-stimulating factors (CSFs) are decreased in potency for stimulating proliferation of hematopoietic progenitor cells (HPCs). T-CXCL12, a modified chemokine, is inactive as an HPC chemotactic, survival, and enhancing factor for replating or ex-vivo expansion of HPCs. Moreover, T-CSFs and T-CXCL12 specifically downmodulates the positively acting effects of their own full-length molecule. Other chemokines have DPP4 truncation sites. In the present study, we evaluated effects of DPP4 inhibition (by Diprotin A) or gene deletion of HPC on chemokine inhibition of multicytokine-stimulated HPC, and on chemokine-enhancing effects on single CSF-stimulated HPC proliferation, as well as effects of DPP4 treatment of a number of chemokines. Myelosuppressive effects of chemokines with, but not without, a DPP4 truncation site were greatly enhanced in inhibitory potency by pretreating target bone marrow (BM) cells with Diprotin A, or by assaying their activity on dpp4/cd26(-/-) BM cells. DPP4 treatment of myelosuppressive chemokines containing a DPP4 truncation site produced a nonmyelosuppressive molecule, but one which had the capacity to block suppression by that unmodified chemokine both in vitro and in vivo. Additionally, DPP4 treatment ablated the single cytokine-stimulated HPC-enhancing activity of CCL3/MIP-1α and CCL4/MIP-1β, and blocked the enhancing activity of each unmodified molecule, in vitro and in vivo. These results highlight the functional posttranslational modulating effects of DPP4 on chemokine activities, and information offering additional biological insight into chemokine regulation of hematopoiesis.Item Nonmarrow hematopoiesis occurs in a hyaluronic-acid-rich node and duct system in mice(Mary Ann Liebert, 2014-11-01) Hwang, Sunhee; Lee, Seung J.; Park, Sang H.; Chitteti, Brahmananda R.; Srour, Edward F.; Cooper, Scott; Hangoc, Giao; Broxmeyer, Hal E.; Kwon, Byoung S.; Department of Medicine, IU School of MedicineA hyaluronic-acid-rich node and duct system (HAR-NDS) was found on the surface of internal organs of mice, and inside their blood and lymph vessels. The nodes (HAR-Ns) were filled with immune cells of the innate system and were especially enriched with mast cells and histiocytes. They also contained hematopoietic progenitor cells (HPCs), such as granulocyte-macrophage, erythroid, multipotential progenitors, and mast cell progenitors (MCPs). MCPs were the most abundant among the HPCs in HAR-Ns. Their frequency was fivefold higher than that of the MCPs in bone marrow. In addition, the system contained pluripotent stem cells (PSCs) capable of producing CD45(-)Flk1(+) hemangioblast-like cells, which subsequently generated various types of HPCs and differentiated blood cells. Although HAR-Ns did not appear to harbor enough number of cells capable of long-term reconstitution or short-term radioprotection of lethally irradiated recipients, bone marrow cells were able to engraft in the HAR-NDS and reconstitute hematopoietic potentials of the system. PSCs and HPCs were consistently found in intravenous, intralymphatic, and intestinal HAR-ND. We infer that PSCs and HPCs reside in the HAR-ND and that this novel system may serve as an alternative means to traffic immature and mature blood cells throughout the body.