bluuebunny/crossref_metadata_2025_split huggingface dataset:
Identification of RIZ1 Targets Involvedin Erythroid Differentiation of K562 Human Erythroleukemia Cells Using Surface-Enhanced Laser Desorption Ionization Time-Of-Flight Mass Spectrometry (SELDI).
Authors: Takahashi Naoto , Brainbridge Matthew , Scott Stuart A. , Ichinohasama Ryo , Sanche Stephen E. , Dong Wei-Feng , DeCoteau John F. , Geyer C. R. 
Published: October 2019

Abstract
RIZ1 (PRDM2) is a member of the nuclear protein methyltransferase superfamily involved in chromatin remodeling. RIZ1 functions as a tumor suppressor gene in a number of human cancers and is down regulated in some human acute myeloid leukemias. We previously found RIZ-1 to be silenced in K562 erythroleukemia cells by promoter hypermethylation. Furthermore, expression of RIZ1 in K562 promotes erythroid differentiation and also potentiates TGF-β1 mediated differentiation. To investigate similarities between genes altered by RIZ1 expression and the TGF-β1 pathway, we used SELDI to compare the protein profiles of K562 against K562 + RIZ1 and K562 + TGF-β1. Protein extracts for SELDI profiling were separated into six fractions according to their isoelectric points. Proteins from each fraction were then bound to two different protein chip surfaces (H50-hydrophoboic and CM10-cation exhange) and their mass/charge determined using SELDI. We analyzed four replicates from each sample and classified proteins as differentially expressed if their P-values were below 0.05. In total, we observed 104 differentially expressed proteins (60 upregulated and 44 down regulated) between K562 and K562 + RIZ1 and 176 proteins (96 upregulated and 80 down regulated) between K562 and K562 + TGF-β1. We used 2D-PAGE to identify differentially expressed proteins identified by SELDI analysis and located 48 proteins that were over expressed in K562 + RIZ1 and K562 + TGF-β1 relative to K562. To establish whether these proteins were the same proteins observed using SELDI, we determined if the proteins had the same pI and molecular weight and if the gel-eluted proteins bound to the same protein chip surface with the same mass/charge. 15 of 48 proteins passed the above criteria and we determined their identities using Trypsin-based peptide mapping strategies with molecular weight and pI restrictions. We identified two candidate proteins (14-3-3ε and S100/A13) that are similarly over expressed in K562 + RIZ1 and K562 + TGF-β1. These proteins have been shown to be associated with TGF-β1 signaling. Schistosomal 14-3-3ε interacts with SmRK1, a divergent type I transforming growth factor β1 receptor (TR-I) present on the surface of adult parasites and also binds to and activates human TR-I. S100/A13 belongs to a family of low molecular weight proteins characterized by the presence of two calcium-binding EF-hand motifs that includes S100C/A11, a member recently shown to play a key role in a PKCα mediated pathway essential for the growth inhibition of normal human keratinocytes by TGF-β1. In summary, we demonstrate the potential for using SELDI to identify novel proteins involved in regulating and connecting cellular growth and differentiation pathways.
Intra-S-Phase Regulator cdc7 Is a Gene Target of the c-Myb Proto-Oncogene in Human Leukemias.
Authors: Shetzline Susan E. , Hsu June , Thalheim Lindsay , Choi John K. , Gewirtz Alan M. 
Published: October 2019

Abstract
The c-myb proto-oncogene encodes a transcription factor, Myb, which is essential for normal hematopoiesis. Myb may also play a role in leukemogenesis but possible mechanisms remain ill defined. To gain further insights into this issue, we sought to identify Myb regulated genes in human myeloid leukemic cells utilizing a tamoxifen inducible expression system and a microarray approach. Myb function was conditionally abrogated by tamoxifen following infection of K562 cells with a bicistronic retroviral vector MIGR1 which had c-myb’s DNA binding domain (DBD) and the Drosophila engrailed protein transcription repressor domain (MERT) fused to a modified estrogen receptor that binds tamoxifen. MERT was subcloned upstream of an IRES-EGFP cassette in MIGR1 to allow for FACS purification on the basis of GFP expression. To identify Myb regulated genes, purified K562-MERT cells were exposed to 1 μM tamoxifen or ethanol (control) for three days, processed for hybridization to the microarray gene chip and analyzed by software algorithms from Incyte and Arrayex. When endogenous Myb activity was suppressed by MERT, 105 genes out of 10,000 genes on the microarray chip changed > 2-fold in expression. Of these 105 genes, 34 increased their expression >2-fold while 70 decreased their expression >2-fold. Since Myb expression is elevated in leukemic cells, we hypothesized that Myb functions in malignant hematopoietic cells to induce the expression of genes that are essential for their maintenance and survival. Therefore, we focused on those genes that decreased in expression when Myb activity was inhibited by MERT. Among the most repressed was cdc7 (2.8-fold decrease), an intra-S-phase regulator. To verify the microarray data, we utilized real-time PCR to quantitate the expression of cdc7 in our K562-MERT cells. Cdc7 expression decreased 5-fold in tamoxifen treated K562-MERT cells relative to control cells, which is consistent with the microarray data. We then performed chromatin immunoprecipitation (ChIP) experiments to determine whether cdc7 is a direct target of Myb. When the chromatin from untreated K562 and K562-MERT cells was immunoprecipitated with anti-c-Myb, we observed one PCR product using a primer pair that flanked the Myb binding sites in the promoter region of cdc7. This same result was observed in our positive control ChIP experiment in which the chromatin was immunoprecipitated with anti-acetyl histone H4, indicating that the region of the cdc7 promoter containing the Myb binding sites is poised for transcription. When Myb transactivation activity was inhibited by MERT in K562-MERT cells, no PCR product was observed following chromatin immunoprepitation with anti-c-Myb. These results strongly suggest that cdc7 is a direct gene target of c-Myb in malignant hematopoietic cells. Investigation of the transcriptional regulation of cdc7 in hematopoietic cells may yield new clues to Myb’s role in leukemogenesis.
Genes Located within the del(9q) Commonly Deleted Region Are Down-Regualted in Acute Myeloid Leukaemia.
Authors: Zhao Zheng , Daly Sarah , Yin John Liu , Sweetser David , Boultwood Jacqueline , Wainscoat James , Peniket Andrew 
Published: October 2019

Abstract
Deletions of 9q are recurring cytogenetic abnormalities in acute myeloid leukaemia (AML). In approximately one-third of cases del(9q) occurs in association with t(8;21). We have previously identified a 2.4Mb region located on 9q21.32–21.33 which is deleted in cases of del(9q) AML - the del(9q) commonly deleted region (CDR). This region encodes 11 genes which we have also previously shown not to be mutated in del(9q) AML. In order to further investigate the role of these genes in AML and in particular to elucidate the pathogenesis of del(9q) AML we have examined the expression of these genes in AML. RNA was extracted from the bone marrow or peripheral blood of patients with AML at the time of diagnosis. Patient samples from the following cytogenetic subgroups were included in this study:
(1) del(9q) AML (n=8) - this includes 3 patients with associated t(8;21); (2) t(8;21) but no del(9q) (n=15); (3) Normal karyotype (n=6); (4) Complex Karyotype (n=6). Taqman assays were designed for 9 of the 11 genes located within the del(9q) CDR: FRMD3; ENSG00000148057; UBQLN1; GKAP42; Q9UF54; Q8N2B1; Q9H9A7; SLC28A3; NTRK2. For the other 2 genes within the region Taqman assays could not be performed because of uniformly low expression levels (Q8IZ41) and lack of specificity of primer-design (HNRPK). CD34-purified progenitors from normal individuals were used as controls.
It was found that 6 of the 9 genes were significantly down-regulated in del(9q) AML (p<0.05): ENSG00000148057; UBQLN1; Q9UF54; Q8N2B1; Q9H9A7; NTRK2. Since del(9q) is commonly associated with t(8;21), cases of t(8;21) in which del(9q) was not present were also analysed for the expression levels of the del(9q) CDR genes. It was found that 5 of the 9 genes were significantly down regulated in t(8;21) AML (ENSG00000148057; Q9UF54; Q8N2B1; Q9H9A7; SLC28A3) (p<0.05). Only two of these genes were found to be down-regulated in AML of normal karyotype (Q9H9A7 and UBQLN1) (p<0.05) and no significant down-regulation was detected in any of these genes in AML of complex karyotype. Our findings indicate that several genes from within the del(9q) AML CDR are down-regulated in del(9q) AML. A similar pattern of down-regulation is found in cases of t(8;21) even in the absence of del(9q) AML. This suggests that down-regulation of one or more of these genes may be important in the pathogenesis of AML. It may therefore be hypothesized that this pattern of gene down-regulation provides a mechanism common to the development of AML with both del(9q) and t(8;21).
Silencing of Caspase 8 Expression in Leukemia Cells and Patient Samples.
Authors: Disperati Patricia , Suarez-Saiz Fernando , Gronda Marcela , Minden Mark D. , Schimmer Aaron 
Published: October 2019

Abstract
Gene silencing through hypermethylation is common in cancer cells and may contribute to chemoresistance and poor patient outcomes. To investigate this mechanism in leukemia, OCI-AML2 leukemia cells were screened for methylated genes. Among the genes identified using Cpg arrays was caspase-8. Levels of mRNA and protein of caspase-8 were decreased in OCI-AML2 cells compared to control cell lines (Jurkat cells). In contrast, levels of FLIP, caspase-3 and XIAP did not differ between both cell lines. As further evidence of caspase-8 hypermethylation, treatment of OCI-AML2 cells with the demethylating agent 5-Aza-2′-Deoxycytidine (AzaD) increased expression of caspase-8 protein to levels similar to Jurkat cells. To determine whether the silencing of caspase-8 occurs in primary patient samples, levels of caspase-8 were measured by immunoblots in 16 samples from patients with AML. Compared to normal peripheral blood mononuclear stem cells, caspase-8 was reduced in five of 16 (31%) samples. To determine whether the low caspase 8 was a functional consequence, OCI-AML2 cells were treated with CH-11 anti-FAS antibody (100 ng/mL) for 24 hours. Despite the cells expressing the FAS receptor on the surface, less than 5% of apoptosis occurred. In contrast, an equal concentration of CH-11 induced 80% apoptosis in Jurkat cells. To test the status of the death receptor pathway downstream of caspase-8, cytosolic lysates from OCI-AML2 cells were stimulated with recombinant active caspase-8 to directly activate effector caspases. Stimulation of OCI-AML2 lysates with recombinant active caspase-8 increased the maximal rate of effector caspase hydrolysis of Ac-DEVD-AFC 8 fold above buffer treated control, comparable to treatment of Jurkat lysates. These results indicate that OCI-AML2 cells have defects above the level of the effector casapses that renders them resistant to death receptor ligands. To determine if silencing of casp-8 is sufficient to explain these defects, OCI-AML2 cells were cultured with AzaD and CH-11 anti-FAS antibody. Despite restoration of caspase-8 protein levels, AzaD did not restore sensitivity to CH-11. Likewise, transfection of caspase-8 into OCI-AML2 did not restore sensitivity. Therefore, casp-8 is silenced in OCI-AML2 cells and primary patient samples. However, the events indicate the presence of additional defects in the death receptor pathway that cannot be reversed by demethylation. These results suggest that blockade of apoptosis throughout the death receptor pathway may occur at several points and that in some cases, simple reversal of demethylation may not be sufficient.
A Role for IL-1Receptor Antagonist in the Acute Therapeutic Effects of IVIg?.
Authors: Crow Andrew R. , Song Seng , Freedman John , Semple John W. , Lazarus Alan H. 
Published: October 2019

Abstract
It has been suggested that IVIg and anti-D may function through the regulation of various cytokines; in particular, significant increases in the anti-inflammatory compound interleukin-1 receptor antagonist (IL-1Ra) have been reported after exposure of patients to IVIg or anti-D. However, the possible role that IL-1Ra may play in the acute therapeutic effects of IVIg or anti-D is unknown. Using a murine model of ITP in which IVIg and anti-RBC-specific antibodies are therapeutically effective, we observed that mice injected with therapeutic doses of IVIg and one anti-RBC antibody (TER-119, which mimics the effects of anti-D) demonstrated increased serum levels of IL-1Ra, reaching peak levels at a time which coincided with an increase in platelet count. Surprisingly, another RBC-specific antibody which also reverses ITP, failed to increase IL-production above basal levels. Thrombocytopenic mice lacking the IL-1 receptor are known to be completely unresponsive to IL-1Ra. These mice responded to both IVIg and TER-119 as successfully as wild-type mice. Injection of mice with as much as 1 mg of recombinant IL-1Ra did not significantly ameliorate thrombocytopenia. To further study the possible indirect role of the IL-1Ra system in the amelioration of murine ITP, we next looked at nitric oxide (NO), an immunoregulatory compound that can be induced by IL-1, stimulate increases in IL-1Ra levels, and has been previously demonstrated to be modulated by IVIg. We report here that treatment of mice with aminoguanidine, an iNOS-selective inhibitor or with L-NAME, a multi-spectrum NOS inhibitor, did not significantly affect the ability of IVIg to ameliorate ITP. These data suggest that while IVIg and TER-119 may mediate the release of IL-1Ra, it is not required for IVIg or anti-RBC antibodies to exert their acute therapeutic effects.
Thrombocytopenia Is a Strong Predictor of All-Cause and AIDS-Specific Mortality in Women with HIV: The Women’s Interagency HIV Study.
Authors: Pearce C. Leigh , Mack Wendy J. , Levine Alexandra M. , Gravink Jay , Cohen Mardge H. , Machtinger Edward L. , Anastos Kathryn , Silverberg Michael J. , Young Mary A. , Minkoff Howard , Watts D. Heather 
Published: October 2019

Abstract
Background: Thrombocytopenia is a common condition among HIV-infected individuals, however its significance is unclear, particularly among women. Two previous studies, one consisting mostly of men (Sullivan PS, et al. J Acquir Immune Defic Syndr.1997;14:374–379) and one of hemophiliacs (Ehmann WC, et al. Am J Hematol.1997; 54:296–300), have suggested that low platelet count is associated with decreased survival.
Methods: The Women’s Interagency HIV Study (WIHS) is a long-term prospective cohort study of HIV-infected women and HIV-negative women that is being conducted at six urban sites across the United States. 1,990 HIV-infected women and 553 HIV-negative women are included in this report. These women are seen every six months; the median follow-up time is 7.5 years. We conducted extensive multivariate analysis using both generalized estimating equations and Cox proportional hazards models in order to determine the predictors of thrombocytopenia and the role of platelet count in mortality among women being followed as part of this study.
Results: At baseline, 15% of HIV-positive women were thrombocytopenic versus 1.6% of HIV-negative women (p<0.001). Factors associated with increased risk of thrombocytopenia included HIV infection, low CD4 cells, increasing viral load, and smoking. African-American women were significantly protected against thrombocytopenia when compared to Whites, as reported by others (Sloand EM, et al. Eur J Haematol. 1992; 48:168–72; Sullivan PS, et al. J Acquir Immune Defic Syndr.1997;14:374–379 ). Resolution of thrombocytopenia was associated with highly-active antiretroviral therapy (p<0.001), especially that containing zidovudine (<0.0001). On multivariate analysis, thrombocytopenia was a significant predictor of mortality, with women having a platelet count <50,000 cells/mm3 being at more than 5-fold increased risk of dying due to any cause, and at 3-fold increased risk of death due to AIDS compared to women with a platelet count in the normal range. Only CD4+ lymphocyte count <200 cells/mm3 was similar in the magnitude of its effect on mortality. The reasons for decreased survival associated with low platelet count in the context of HIV-infection are unclear and further study is needed.
Conclusions: (1) Thrombocytopenia is associated with HIV infection (p<0.001), and with parameters of more advanced HIV disease in women; (2) African American HIV + women are protected from thrombocytopenia compared to HIV + white women (p<0.0001); (3) HAART is associated with resolution of thrombocytopenia, especially those regimens including AZT (p<0.001); (4) Thrombocytopenia is an independent risk factor for decreased survival in HIV infected women.
Iron Containing Nucleosides as New Cytostatic Substances for Cancer and Leukemia Therapy, Especially for Therapy of Relapsed ALL in Childhoodtransfusion of Red Blood Cells under Shock Conditions in the Rat Microvasculature.
Authors: Prokop Aram , Wieder Thomas , Schlawe Daniel , Lode Holger , Henze Guenter , Daniel Peter T. , Schmalz Hans G. 
Published: October 2019

Abstract
Acute lymphoblastic leukemia (ALL) is the most common malignant disease in childhood. Despite a relatively good prognosis approximately one fourth of the patients suffers from relapse and, consequently, a worse prognosis. Only 40 % of these children with relapsed ALL will survive. Patients who fail to achieve a complete remission in chemotherapy do not survive. This explains the need for new compounds that may overcome resistance against clinically employed cytostatic drugs. Here we describe a new class of iron-containing nucleosides that overcome drug resistance in vitro and ex vivo. Analyses of apoptosis induction in primary lymphoblasts from 30 children with de novo ALL and relapsed ALL showed that these iron-containing nucleoside analogs are far more potent as compared with conventional nucleoside analogs employed in clinical therapy of childhood ALL (cytosinarabinoside: p < 0.0064). Furthermore we demonstrate ex vivo these novel nucleoside analogs overcome drug resistance against fludarabine (p<0.0162) and doxorubicin (p<0.002).
Additional experiments revealed that the prototype of iron-containing nucleoside analogs, N69, specifically induced apoptosis, as evidenced by DNA fragmentation, dissipation of the mitochondrial membrane potential and activation of caspase-3. All those events occurred in the absence of cellular lysis, i. e. in the absence of release of lactate dehydrogenase from the cells, thereby excluding N69-induced necrosis.
N69-induced cell death was functionally characterized by the use of different cellular model systems being devoid of defined molecular parts of the apoptosis machinery. According to those results, the CD95-dependent apoptosis cascade is not involved in N69-induced cell death. Furthermore, N69 triggers apoptosis in a Bax-independent manner in the essentially Bax-free prostata carcinoma cell line DU145. Thus, N69 is able to break the Bax-related drug resistance that plays an important role in the therapy of relapsed ALL in childhood. In addition, we could show that N69-induced apoptosis even proceeds in the absence of caspase-3 expression. We demonstrate here that N69 still exerts a considerable apoptosis-inducing effect in the caspase-3-deficient, multi drug-resistant (i. e. taxol-, epirubicin- and etoposide-resistant) mamma carcinoma cell line MCF-7. Taken together, iron-containing nucleosides comprise a totally new, very promising class of cytostatic agents for cancer and leukemia therapy, especially for the therapy of relapsed ALL in childhood. Future studies will aim to elucidate the target structures of these new compounds and to demonstrate N69-mediated anti-cancer activity in vivo. First experiments in mice show a good tolerability of N69.
In Vivo Bone Marrow Stem Cell Gene Transfer in Mice by In Situ Delivery of a 3rd-Generation Lentiviral Vector Using Intrafemoral Injection.
Authors: Pan Dao , von Kalle Christof , Williams David A. , Worsham D. Nicole , Bohn Kimberly , Lutz Christopher 
Published: October 2019

Abstract
HIV-based lentiviral vectors (LV) have become powerful tools for in vivo gene transfer and gene expression in non-dividing cells after local injection. However, the potential of in vivo bone marrow stem cell gene transfer by “in situ” bone cavity injection has not been well unexplored. This in vivo approach could take full advantage of any source of stem cells present in bone cavity and avoid many of the difficulties encountered by ex vivo hematopoietic stem cell (HSC) gene transfer. To determine if intra bone marrow (iBM) injection of LV can efficiently transduce BM stem cells, a 3rd-generation self-inactivating LV, containing eGFP regulated transcriptionally by a CMV promoter, was used to inject intra-femorally into adult Boy/J Ly45.1 mice (at 2 x106 IU/mouse). Blood counts were normal in all LV-injected mice (n=4) and buffer-injected mice (n=4). GFP-expressing peripheral blood leukocytes (PBL) were observed in both myeloid (up to 4%) and lymphoid subpopulations (up to 2%) 3-month post injection. To evaluate gene transfer and transgene expression in multi-potential progenitors, the colony-forming cell (CFC) assay was performed using low-density bone marrow 4-months post injection, resulting in 4–5% GFP+ CFU-granulocyte macrophage (CFU-GM) and CFU-granulocyte, erythroid, macrophage and megakaryocyte (CFU-GEMM). This was consistent with the 4-color FACS analysis data demonstrating that up to 3.7% of HSC/progenitors (Lin−c-kit+Sca1+ cells) from LV-injected mice expressed GFP transgene 4-months post injection. To further evaluate HSC gene transfer, BM from injected mice was transplanted into lethally irradiated adult C57/Bl6 (Ly45.2) mice. Four months later, successful engraftment was demonstrated in all BM recipients with 97–99% donor-derived cells in PBL and 91–93.3% in BM. Moreover, significant levels of GFP+ CFU were observed in all recipients ranging from 8.4 ± 2.3% to 17.7 ± 4.2%. To further study the LV-mediated gene transfer profile in HSC, mesenchymal progenitors and systemic distribution by intra-femoral injection, molecular analyses are in progress including real-time QPCR and clonality analysis by LAM-PCR. Our results indicate successful LV-mediated gene transfer into HSC by iBM injection. These data would potentially open a door to a novel approach for treatment of human diseases, but also provide a new tool to study adult stem cell plasticity and the nature of hematopoiesis.