bluuebunny/crossref_metadata_2025_split huggingface dataset:
WP-1066, a Next-Generation Member of JAK-Stat Inhibitors, Induces Cell Cycle Arrest, Abrogates Proliferation, and Induces Apoptosis of Acute Myeloid Leukemia (AML) Cells.
Authors: Ferrajoli Alessandra , Faderl Stefan , Van Quin , Harris David M. , Priebe Waldemar , Kantarjian Hagop M. , Talpaz Moshe , Estrov Zeev 
Published: April 2020

Abstract
Janus kinases (JAK) are tyrosine kinases associated with both cytokine receptors and downstream signal transducer and activator of transcription (Stat) proteins. Upon activation of JAK by a variety of cytokines and growth factors, Stats translocate to the nucleus and promote transcription of target genes. Constitutive activation of Stat proteins in AML has been associated with poor prognosis and AG490, an inhibitor of this pathway, was shown to suppress AML cell proliferation in vitro. WP-1066 represents a further development of AG490 with biological activity at significantly lower concentrations. Therefore, we studied the effects of WP-1066 on the AML cell lines OCIM2 and K562 and on fresh bone marrow aspirates obtained from five newly diagnosed AML patients. We found that WP-1066 inhibited the proliferation of OCIM2 and K562 cells and of fresh marrow AML blast colony-forming cells in a dose-dependent fashion at concentrations ranging from 0.5 to 3 μM. WP-1066 completely abrogated the growth of leukemia cells at a concentration of 3 μM. Furthermore, WP-1066 induced a cell cycle arrest of OCIM2 and K562 cells. Incubation of AML cells with 2 μM of WP-1066 resulted in a time-dependent accumulation of OCIM2 and K562 cells in the sub-G0 phase of the cell cycle. Those leukemia cells underwent apoptotic cell death as assessed by annexin V-FITC. Incubation of OCIM2 cells with 0.5 to 3 μM WP-1066 for 2 hours induced a dose-dependent apoptosis in 52% of the cells. A 4 hour exposure of either OCIM2 or K562 cells to 2 μM of WP-1066 induced caspase 3 activation and PARP cleavage. As expected, WP-1066 inhibited Stat3 and Stat5 phosphorylation in K562 and OCIM2 cells both in a time- and dose-dependent manner, confirming that inhibition of the JAK-Stat pathway is its mechanism of action. Overall, our data showing that WP-1066 inhibits the JAK-Stat pathway, suppresses proliferation, induces cell cycle arrest and apoptosis of AML cells, suggest that the activity of this compound warrants further exploitation aimed at developing WP-1066 for future therapy of AML.
Cyclosporine A Modulates the Multidrug Resistance Proteins P-Glycoprotein, Multidrug Resistance Protein, Breast Cancer Resistance Protein and Lung Resistance Protein.
Authors: Qadir Misbah , O’Loughlin Kieran L. , Williamson Nicole A. , Minderman Hans , Baer Maria R. 
Published: April 2020

Abstract
Multidrug resistance (MDR) mediated by the ATP-binding cassette proteins P-glycoprotein (Pgp), multidrug resistance protein (MRP-1), breast cancer resistance protein (BCRP) and the vault protein lung resistance protein (LRP) is implicated in treatment failure in acute myeloid leukemia (AML). Pgp, MRP-1 and BCRP mediate energy-dependent cellular drug efflux, while LRP blocks cytoplasmic-nuclear drug transport. MDR modulators are non-cytotoxic drugs that block the activity of MDR proteins. In clinical trials, the Pgp modulator cyclosporine A (CsA) improved treatment outcome in AML, while its non-immunosuppressive, non-nephrotoxic analogue PSC-833 has not, despite being a potent Pgp modulator in preclinical models. CsA is known to also modulate MRP-1, and we hypothesized that a broad spectrum of MDR modulation might contribute to its clinical efficacy. We studied the effects of CsA and PSC-833 on in vitro drug uptake, retention and cytotoxicity in drug-selected and transfected resistant cell lines overexpressing Pgp, MRP-1 or BCRP, and on nuclear-cytoplasmic drug distribution and cytotoxicity in cell lines overexpressing LRP. Cellular drug content was measured by flow cytometry, and nuclear-cytoplasmic drug distribution was assessed by confocal microscopy. CsA enhanced uptake and retention of the substrate drug mitoxantrone in cells overexpressing Pgp (HL60/VCR), MRP-1 (HL60/ADR) and BCRP (8226/MR20) and increased cytotoxicity 7-, 4- and 4-fold, respectively. Moreover, CsA increased the nuclear content of doxorubicin in 8226/MR20 cells, which co-express LRP with BCRP, and increased doxorubicin cytotoxicity 12-fold. The effect of CsA on nuclear drug content and cytotoxicity in 8226/MR20 cells occurred without an effect on cellular doxorubicin content, consistent with the fact that 8226/MR20 cells co-express wild type BCRP (BCRPR482), which does not efflux doxorubicin. Moreover the BCRP modulator fumitremorgin C had no effect on 8226/MR20 content, nuclear uptake nor cytotoxicity of doxorubicin. CsA also enhanced nuclear doxorubicin content in a second cell line with LRP-mediated resistance, HT1080/DR4. PSC-833 enhanced mitoxantrone retention and cytotoxicity in cells overexpressing Pgp, but, in contrast to CsA, had no effect on mitoxantrone uptake, retention nor cytotoxicity in cells expressing MRP-1 nor BCRP, and had no effect on doxorubicin nuclear content nor cytotoxicity in cells expressing LRP. Thus CsA is a broad-spectrum MDR modulator, with activity against Pgp, MRP-1, BCRP and LRP, while PSC-833 only modulates Pgp. The broad-spectrum activity of CsA may contribute to its clinical efficacy. These findings support identification and testing of other broad-spectrum MDR modulators.
Murine Mesenchymal Stem Cells Improve Haemopoietic Engraftment in a Murine Transplant Model and Is Maximised through Osteogenic Stimulation.
Authors: Grainger John D. , Fairbairn Lez J. , Wynn Robert F. 
Published: April 2020

Abstract
Several studies have preciously shown improved engraftment of human haemopoietic stem cells (HSC) following co-infusion of human mesenchymal stem cells (MSC). However, these studies have been criticised for using xenogeneic recipients which might support human haemopoiesis through the production of species-specific cytokines and growth factors by human MSC. To further investigate the potential for MSC to support HSC engraftment we used a murine to murine transplant model to co-infuse purified murine MSC and murine HSC.
The MSC were derived from collagenase-treated bone fragments of the Rosa26 murine strain, had the phenotype CD45- CD31-CD11b- Sca-1bright VCAM1+ and could differentiate into osteocytes, chondrocytes and adipocytes. Donor and recipient HSC were obtained from unmanipulated bone marrow of either the C57Bl/6J murine strain, CD45 isotype CD45.2, or the PEP3b murine strain, expressing CD45.1. Following 10Gy irradiation to the recipients HSC and MSC were co-infused. Donor and recipient haemopoietic engraftment in the peripheral blood was quantified by FACS analysis of the CD45.1/CD45.2 ratio.
Successive transplant experiments persistently showed improved donor haemopoietic engraftment following co-infusion of 2 x 105 BM cells and 1 x 106 MSC (three seperate transplant series p=0.001, p=0.09 and p=0.13). The beneficial effect was maximal at the lower HSC dose of 1 x 105 BM cells (p=0.004, p=0.045 and uninterpretable in the third transplant series due to excessive death observed in the non-MSC recipients).
Increasing the MSC dose to 2 x 106 cells showed a non-significant increase in survival and donor haemopoietic engraftment but further MSC escalation resulted in fatal emboli post-infusion.
LacZ staining of tissue sections failed to show evidence of MSC outside the lungs but was detected at very low levels in the bone from one recipient.
As MSC are osteoblast precursors and recent literature suggests a role of osteoblasts as the HSC niche we investigated the effect of pre-culturing our MSC population in osteogenic media prior to transplant. Following 16 days of culture approximately 40% of colonies showed ALP activity. At this time point osteogenic-stimulated MSC (O-MSC) were removed from culture, washed to remove any residual osteogenic media, and infused in our standard method. O-MSC recipients receiving 1 x 105 BM cells showed a mean engraftment of 79% compared to 21% in non-msc recipients receiving 2 x 105 BM cells (p=0.000017), and 42% in recipients receiving standard MSC and 1 x 105 BM cells (p=0.01).
In conclusion we confirm improved haemopoietic engraftment in a non-xenogeneic model and does not require significant MSC engraftment. The beneficial effect is maximised through osteogenic stimulation of MSC suggesting the possibility that the mechanism may be through priming of the HSC niche.
Human Memory T Cells Proliferate but Do Not Elicit Cytotoxicity in Response to Alloantigens.
Authors: Chen Benny J. , Cui Xiuyu , Chao Nelson J. 
Published: April 2020

Abstract
We and others have recently demonstrated that memory T cells do not induce graft-versus-host disease in several different animal models. To test whether the same concept applies to humans, we compared the ability of memory T cells to respond to alloantigens with that of naive as well as bulk T cells. Purified T cells were first obtained from peripheral blood from healthy donors and then separated into memory and naive T cell subsets based on the expression of CD45RA (memory: CD45RA−, naive: CD45RA+). Memory T cells were subsequently tested for their ability to respond to alloantigens using proliferation and cytotoxicity assays in comparison with naive and bulk T cells. Proliferation assay was performed using 1.25x105 responder cells and 5x105 irradiated stimulator cells per well in 96-well flat-bottom plate. Cytotoxicity was measured by the standard 4-hour Cr-51 release assay after 5-day mixed lymphocyte culture. In contrast to the mouse data, memory T cells proliferated equally well as naive and bulk T cells did in mixed lymphocyte culture (Figure A). However, these same memory T cells failed to kill the allogeneic targets despite the vigorous proliferative responses against the same alloantigens (Figure B). These data demonstrated that human memory T cells proliferate but do not elicit cytotoxicity in response to alloantigens, suggesting that human memory T cells may not contain true alloantigen-specific T cells if they have never exposed to those alloantigens before and may not cause graft-versus-host disease upon in vivo transfer. These observations warrant the further testing of human memory T cells in clinically more relevant models and in vivo for their ability to induce graft-versus-host disease.
Figure Figure
Dominant T-Cell Clonal Expansion Associated with Acute Graft-Versus-Host Disease after Allogeneic Hematopoietic Stem Cell Transplantation in Pediatric Patients.
Authors: Chen Xiaohua , Knowles James , Barfield Raymond , Lawrence Dawn , Handgretinger Rupert 
Published: April 2020

Abstract
Allogeneic hematopoietic stem cell transplantation (AHSCT) provides a valuable therapy for a variety of malignancies, solid tumors, hematological disorders and autoimmune diseases. One of the major complications in recipients after transplantation is graft-versus-host disease (GvHD), which can cause delayed T-cell reconstitution and therefore increase the risk of infection and mortality. It has been suggested that T cells play an important role in the development of GvHD. Several previous studies have identified T-cell clones associated with acute GvHD in adult patients. However, T-cell clonal expansion in the early stage of post-transplantation might also be caused by T-cells which are unrelated to acute GvHD. In this study, we used TCRβ CDR3 size spectratyping to detect T-cell clonal expansion among the polyclonal complexities of peripheral blood. We examined the TCRβ repertoire distribution at the onset of acute GvHD in 10 pediatric patients. We then compared that result with the TCRβ pattern at the same post-AHSCT stage in another 10 pediatric patients without acute GvHD.
Method: An RT-PCR assay for Vβ CDR3 size distribution was performed. Each fluorescent PCR product was detected by an automated 310 DNA sequencer. The data were analyzed by GeneScan software.
Results and Discussion: As expected, the TCRβ repertoire distribution was much skewed for all patients in both groups, because the immune suppression occurred at early stage of post-AHSCT. There were about 4–8 T-cell clones expanded in each patient for both groups. The TCRβ subfamilies among those clones varied significantly from patient to patient. However, Vβ2, Vβ9, Vβ13 and Vβ20 were commonly found in both groups, indicating in these cases that clonal expansion was not related to the occurrence of acute GvHD. Notably, TCR Vβ16 was found in 9 of 18 samples from 7 of 10 patients with acute GvHD. This TCR subfamily was not dominant in the patients without acute GvHD. This finding strongly suggests that the T-cell clone carrying TCR Vβ16 subfamily might be associated with acute GvHD in pediatric patients. The characterization of this T-cell clone is under study.
Rb Regulates Erythroid Differentiation through Bcl-XL-Dependent Anti-Apoptotic Effect.
Authors: Matsuoka Sahoko , Hirao Atsushi , Arai Fumio , Ito Keisuke , Takubo Keiyo , Nomiyama Kana , Hamaguchi Isao , Ikeda Yasuo , Suda Toshio 
Published: April 2020

Abstract
Inactivation of the retinoblastoma (Rb) gene results in embryonic lethality due to severe anemia and increased nucleated erythrocytes by day E14.5. However, molecular mechanisms of the function of Rb in erythroid differentiation have been unclear. Recent studies have suggested that Rb has both intrinsic and extrinsic roles on erythroid differentiation. Here we showed that Rb regulates terminal erythroid differentiation through inhibition of apoptosis. Enucleation of erythroblasts was impaired in semisolid culture of Rb−/− hematopoietic progenitors in fetal liver. The lethally-irradiated recipient mice transplanted with Rb−/− hematopoietic stem cells (HSCs) showed severe anemia (Hgb 8.2±1.47 g/dl versus WT :12.0±1.22 g/dl) with splenomegaly, whereas the number of leukocytes and platelets were normal. In Rb−/− recipient mice, the nucleated erythrocytes and reticulocytes were significantly increased in the peripheral blood. We analyzed cell surface markers for erythroid lineage (TER119 and CD71) in the enlarged spleen. A block of erythroid differentiation at the early erythroblast stage, accompanied with increased apoptosis, was observed in the recipient mice with Rb−/− HSCs. We speculated that the defect in the erythroid differentiation of Rb−/− HSCs might be caused by inappropriate cell death. Thus, we examined expression profiling for apoptosis-related genes in early erythroblasts (CD71highTer119high) by RT-PCR. Interestingly, the dramatic decrease of Bcl-XL expression was detected in the erythroblasts, whereas other Bcl-2 family members including Bcl-2 and Bax were unchanged. To clarify the function of Bcl-XL, we introduced exogenous cDNA of mouse Bcl-XL with GFP (Bcl-XL ires GFP) or GFP alone as control into HSCs and then transplanted them to lethally irradiated mice. From the point of CD71 and Ter119 expression pattern in GFP positive cells, Rb−/− erythoblasts still showed the block in differentiation. In contrast, a ratio of late erythroblasts in Bcl-XL expressing cells was higher than that in GFP control cells in Rb−/− recipient mice and a number of annexin V positive cells were much reduced in the mice transplanted with Bcl-XL expressing cells, indicating that overexpression of Bcl-XL inhibited inappropriated apoptosis and restored the differentiation. These data demonstrates that Rb regulates erythroid differentiation through Bcl-XL-dependent anti-apoptotic effect.
A Role for NIPA in DNA Damage Response.
Authors: von Klitzing Christine , Bassermann Florian , Morris Stephan W. , Peschel Christian , Duyster Justus 
Published: April 2020

Abstract
The nuclear interaction partner of ALK (NIPA) is a nuclear protein identified by our group in a screen for NPM-ALK interaction partners. We recently reported that NIPA is an F-box protein that assembles with SKP1, Cul1 and Roc1 to establish a novel SCF-type E3 ubiquitin ligase. The formation of the SCFNIPA complex is regulated by cell cycle-dependent phosphorylation of NIPA that restricts SCFNIPA assembly from G1- to late S-phase, thus allowing its substrates to be active from late S-phase throughout mitosis.
Proteins involved in cell cycle regulation frequently play a role in DNA damage checkpoints. We therefore sought to determine whether NIPA has a function in the cellular response to genotoxic stress. For this reason we treated NIH/3T3 cells with various DNA-damaging agents. Surprisingly, we observed phosphorylation of NIPA in response to some of these agents, including UV radiation. This phosphorylation was cell cycle phase independent and thus independent of the physiological cell cycle dependent phosphorylation of NIPA. The relevant phosphorylation site is identical to the respective site in the course of cell cycle-dependent phosphorylation of NIPA. Thus, phosphorylation of NIPA upon genotoxic stress would inactivate the SCFNIPA complex in a cell cycle independent manner. Interestingly, this phosphorylation site lies within a consensus site of the Chk1/Chk2 checkpoint kinases. These kinases are central to DNA damage checkpoint signaling. Chk1 is activated by ATR in response to blocked replication forks as they occur after treatment with UV. We performed experiments using the ATM/ATR inhibitor caffeine and the Chk1 inhibitor SB218078 to investigate a potential role of Chk1 in NIPA phosphorylation. Indeed, we found both inhibitors to prevent UV-induced phosphorylation of NIPA. Current experiments applying Chk1 knock-out cells will unravel the role of Chk1 in NIPA phosphorylation. Additional experiments were performed to investigate a function for NIPA in DNA-damage induced apoptosis. In this regard, we observed overexpression of NIPA WT to induce apoptosis in response to UV, whereas no proapoptotic effect was seen with the phosphorylation deficient NIPA mutant. Therefore, the phosphorylated form of NIPA may be involved in apoptotic signaling pathways.
In summary, we present data suggesting a cell cycle independent function for NIPA. This activity is involved in DNA damage response and may be involved in regulating apoptosis upon genotoxic stress.
Interaction of Stem Cells and Their Niche: Behavior and Gene Expression Profiles of CD34+/CD38− Cells upon Co-Cultivation with AFT024.
Authors: Wagner Wolfgang , Saffrich Rainer , Wirkner Ute , Eckstein Volker , Blake Jonathon , Ansorge Alexandra , Schwager Christian , Miesala Katrin , Ansorge Wilhelm , Ho Anthony D. 
Published: April 2020

Abstract
Cell-cell contact between stem cells and cellular determinants of the microenvironment plays an essential role in the regulation of self-renewal and differentiation. The stromal cell line derived from murine fetal liver (AFT024) has been shown to support maintenance of primitive human hematopoietic progenitor cells (HPC) in vitro. We have studied the interaction between HPC (defined as CD34+/CD38− umbilical cord blood cells) and AFT024 and the impact of co-cultivation on the behavior and gene expression of HPC. By time lapse microscopy the mobility and behavior of CD34+/CD38− cells were monitored. Approximately 30% of the CD34+/CD38− cells adhered to the cellular niche through an uropod. CD44 and CD34 were co-localized at the site of contact. Gene expression profiles of CD34+/CD38− cells were then compared upon co-cultivation either with or without AFT024. After cultivation for 16h, 20h, 48h or 72h the HPC were separated form the feeder layer cells by a second FAC-Sort. Differential gene expression was analyzed using our Human Genome cDNA Microarray of over 51,145 ESTs. Among the genes with the highest up-regulation in contact with AFT024 were several genes involved in cell adhesion, proliferation and DNA-modification including tubulin genes, ezrin, complement component 1 q subcomponent 1 (C1QR1), proto-oncogene proteins c-fos and v-fos, proliferating cell nuclear antigen (PCNA), HLA-DR, gamma-glutamyl hydrolase (GGH), minichromosome maintenance deficient 6 (MCM6), uracil-DNA glycolase (UNG) and DNA-methyltransferase 1 (DNMT1). In contrast, genes that were down-regulated after contact with AFT024 included collagenase type iv (MMP2), elastin (ELN) and hemoglobin genes. Differential expression of six genes was confirmed by RT-PCR. Other authors have reported on the differential gene expression profiles of CD34+ cells derived from the bone marrow versus those from G-CSF mobilized blood. As CD34+ cells from the bone marrow might represent cells exposed to the natural HPC niche we have then compared our findings with these experiments. In these comparisons we identified several overlapping genes that are involved in regulation of cell cycle and DNA repair including PCNA, DNMT1, MCM6, MCM2, CDC28 protein kinase regulatory subunit 1B (CKS1B), Topoisomerase II (TOP2a), DNA Ligase 1 (LIG1) and DNA mismatch repair protein MLH1. All these genes were up-regulated among CD34+/CD38− cells upon co-culture with AFT024, as well as among CD34+ cells derived from the bone marrow versus those from peripheral blood. Our studies support the hypothesis that intimate contact and adhesive interaction of HPC with their niche profoundly influenced their proliferative potential and their differentiation program.