Dose Related Changes in Cell Cycle of Bone Marrow and Spleen Cells Monitored by DNA/RNA Flow Cytometry

1984 ◽  
pp. 219-227
Author(s):  
W. G. Eisert ◽  
H. U. Weier ◽  
G. Birk
Keyword(s):  
Cell Cycle ◽  
Bone Marrow ◽  
Flow Cytometry ◽  
Spleen Cells

scholarly journals Increased Expression of HERG1 K+ Channels Contribute to MDS Progression and Display Correlation with Prognosis Stratification

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 5193-5193
Author(s):  
Li Lu ◽  
Wen Du ◽  
Wei Liu ◽  
Dongmei Guo ◽  
Shiang Huang ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Stem Cells ◽  
Bone Marrow ◽  
Flow Cytometry ◽  
Cell Proliferation ◽  
Therapeutic Target ◽  
Mononuclear Cells ◽  
Refractory Anemia ◽  
Potential Therapeutic Target ◽  
K Channels

Abstract Background: Myelodysplastic syndromes (MDS) are defined as a heterogeneous group of clonal hematopoietic stem cell (HSC) malignant disorders which are characterized by bone marrow failure and dysplasia of blood cells. As continually terminated in AML, MDS are treated as the "pre-leukemia" condition. HERG K+ channels, being three subtypes: HERG1, HERG.2, HERG3, are expressed transiently at early stages of cells such as progenitor and stem cells, disappearing at later stages of cells like mature cells. Our previous work and others demonstrated that HERG1, as an oncoprotein, was over expressed in AML cells and played crucial roles in SDF-1induced leukemia cell migration. The expression and functional role of HERG1 K+ channels in MDS development is not reported. We investigated the HERG1 K+ channels expression and explored the functional link between HERG1 K+ channels and MDS progression Methods: The expression of HERG1 K+ channels in untreated MDS, AML patients and normal control was detected by flow cytometry. The roles of HERG1K+ channels in regulation of SKM-1 cell proliferation, apoptosis and cell cycle by CCK-8 assay and flow cytometry, respectively. Results: We observed that the expression of HERG1 K+ channels on bone marrow (BM) mononuclear cells (MNCs) in MDS patients was significantly higher than that in the controls (42 ± 7.62% vs 19.8 ± 2.79%, p < 0.01) , but was lower than that in AML (42 ± 7.62% vs 52.18 ± 9.72%, p < 0.01). MDS subtypes mainly contained refractory cytopenia unilineage dysplasia (RCUD), refractory cytopenia with multilineage dysplasia (RCMD), refractory anemia with excess blast I (RAEB-I), refractory anemia with excess blasts II (RAEB-II) and MDS-unclassified (MDS-U). We next analyzed percentage of HERG1 K+ channels in MDS subtypes and found that level of HERG1 K+ channels on the MNCs in each subtypes of MDS was significant higher than that in the control group (Control: 19.8 ± 2.79%, MDS-RCUD: 30.91 ± 1.48%, MDS-RCMD: 39.06 ± 2.47%, MDS-RAEB-I: 44.76 ± 5.54%, MDS-RAEB-II: 49.69 ± 3.28%, p < 0.01), suggesting that HERG1 K+ channels expression might be positively associated with malignancy degree of MDS. HSCs played important roles in the pathophysiology of MDS. Our results also revealed that with the increase of malignancy degree, the percentage of HERG1K+ channels on CD34+CD38- derived cells from MDS subtypes tended to elevate in corresponding MDS subtypes (MDS-RCUD: 61.21 ± 9.46%, MDS-RCMD: 65.66 ± 4.57%, MDS-RAEB-I: 72.35 ± 9.38%, MDS-RAEB-II: 75.71 ± 4.24%, p < 0.05), which supported the notion in other way that MDS are HSC malignant disorders. The over-expression of HERG1 K+ channels on CD34+CD38- cells in MDS patients might be correlated with the oncogenesis of MDS. In addition, the prognosis stratification of MDS patients was performed according to International Prognostic Scoring System (IPSS) scores and the untreated MDS cohort was categorized as four risk groups: Low-R, Int-1-R, Int-2-R and High-R. Our results showed that there was a positively correlation between HERG1 level and IPSS scores of patients (Low-R: 31.93 ± 3.47%, Int-1-R: 39.95 ± 5.76%, Int-2-R: 45.94 ± 6.34%, High-R: 49.05 ± 3.04%, p < 0.01). This showed that expression level of HERG1 K+ channels was helpful for predicting the prognosis of de novo MDS. Furthermore, we analyzed HERG1 K+ channel role on MDS cell proliferation and apoptosis. Incubation with 0, 10, 20 uM E-4031 ( HERG K+ channels inhibitor) with SKM-1 cells (MDS cell line) for 48 h, the results showed that blockage of HERG1 decreased the proliferation of SKM-1 cells but had rarely effects on cell apoptosis and cell cycle distribution. In consistent with other studies, HERG1 K+ channels had already been shown to be necessary for growth of cancer cells through specific activities independent of cell cycle such as interacting with TNFR1 protein which could activate NF-κB to facilitate cell proliferation and favoring transduction of growth signals by MAP kinase/c-fos pathway. These findings showed that physiological activity of HERG1 K+ channels was crucial for MDS cell proliferation and HERG1 K+ channels may be a potential therapeutic target for MDS. Conclusion: Briefly, our study firstly showed that HERG1 K+ channels were aberrantly over-expressed on MDS stem cells, mononuclear cells, and positively associated with malignancy degree of MDS. HERG1 K+ channels functionally contribute to MDS progression and may be a potential therapeutic target for MDS. Disclosures No relevant conflicts of interest to declare.

scholarly journals Investigating the Role of C/Ebpδ-Deficiency in Radiation-Induced Bone Marrow Failure

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 5103-5103
Author(s):  
Debajyoti Majumdar ◽  
Eric Pietras ◽  
Jason Stumhofer ◽  
Snehalata A Pawar
Keyword(s):  
Cell Cycle ◽  
Bone Marrow ◽  
Flow Cytometry ◽  
Dna Damage ◽  
Bone Marrow Failure ◽  
Time Points ◽  
Post Irradiation ◽  
Radiation Induced ◽  
Myeloid Progenitors

Abstract Background: Bone marrow (BM) failure is a common side-effect of toxicity to the hematopoietic tissue both in the clinical setting as well as in case of total body irradiation (TBI) exposure in the event of nuclear terrorism or disasters. Particularly IR-induced myelosuppression is considered a significant risk factor for infections and increased risk for long-term hematopoietic dysfunction and myelodysplasia. Although a lot is known about the key regulatory proteins of steady-state hematopoiesis, not much is known about the regulatory factors involved in stress-induced hematopoiesis. Therefore delineating the mechanisms underlying the effects of IR stress-induced hematopoiesis is critical for the development of novel interventions with the potential to prevent or alleviate IR-induced BM injury/failure. The transcription factor CCAAT/enhancer-binding protein delta (Cebpd; C/EBPδ) plays an important role in the regulation of inflammatory and stress responses, and in the innate and adaptive immune responses. We have previously reported that Cebpd-knockout (KO) mice display increased neutropenia, thrombocytopenia and myelotoxicity in response to IR exposure, which correlated with increased apoptosis of hematopoietic stem cells (HSCs) and hematopoietic progenitor cells (HPCs). In the present study, we further investigated the underlying mechanisms of IR-induced bone marrow failure in the absence of C/EBPδ. Methods: Cebpd+/+and Cebpd-/- mice (C57BL/6J background) aged 3 months were exposed to 6 Gy TBI. Bone marrow mononuclear cells (BM-MNCs) were isolated from femurs and tibiae harvested at early time-points (1h, 4h and 24h) as well as 2 weeks post-irradiation. The presence of reactive oxygen species (ROS) was measured using MitoSOX and the extent of DNA damage was measured using an antibody specific to g-H2AX at the above time points. BM-MNCs were labelled with fluorophore-tagged antibodies and analyzed by flow cytometry to measure the absolute numbers of long term-HSCs, multipotent progenitors, common lymphoid progenitors and myeloid progenitors at 2 weeks post-6 Gy. The changes in cell cycle distribution in response to IR exposure will also be analyzed by flow cytometry using a DNA-binding dye in conjunction with Ki67. Results: Preliminary results reveal: (a) reduced numbers of HSCs, HPCs, common myeloid progenitors, myeloid-erythroid progenitors and granulocyte-monocyte progenitors and (b) increased accumulation of ROS and the DNA damage marker, γ-H2AX in HSCs and HPCs in Cebpd-/- mice compared to Cebpd+/+ mice at 2 weeks post-irradiation. These results suggest that C/EBPδ may play a protective role in radiation-induced bone marrow injury. Studies are underway to further examine changes in cell cycle, ROS and DNA damage in the various BM cell lineages post-TBI. (Funding support by NIGMS P20GM109005 & Department of Defense W81XWH-15-1-0489 is gratefully acknowledged) Disclosures No relevant conflicts of interest to declare.

GD2+ Selected Mesenchymal Stromal Cells (MSCs) Demonstrate a More Robust Proliferation and Differentiation Potential Compared to Unselected Cells.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 1919-1919
Author(s):  
Caridad Martinez ◽  
Ted J. Hofmann ◽  
Roberta Marino ◽  
Massimo Dominici ◽  
Edwin M. Horwitz
Keyword(s):  
Cell Cycle ◽  
Bone Marrow ◽  
Flow Cytometry ◽  
Mesenchymal Stromal Cells ◽  
Stromal Cells ◽  
Doubling Time ◽  
Blue Staining ◽  
Differentiation Potential ◽  
Proliferation And Differentiation

Abstract Human mesenchymal stromal cells (MSCs) are spindle-shape, plastic-adherent cells with capacity to differentiate to bone, cartilage, and fat. MSCs express fibroblast, endothelial, and lymphocyte antigens, e.g. CD105, CD73, CD90, and CD166 which are the cornerstone of phenotypic characterization of these cells. We recently showed that MSCs are the only bone marrow cell to express GD2, a neural ganglioside. Now, for the first time we show that GD2 may serve as the single, unique, and definitive marker of marrow and adipose derived MSCs that can be used to isolate GD2+ MSCs, which possess important biologic properties justifying prospective isolation. MSCs expression of GD2 is uniformly high on freshly isolated and culture-expanded cells. Using the Miltenyi AutoMACS® device and a monoclonal antibody recognizing GD2 (clone 14.G2A) we prospectively isolated a highly enriched MSC population from bone marrow MNCs. The selected fraction was &gt;98% pure for GD2+ cells determined by flow cytometry. Light microscopy showed that the GD2-selected cells were smaller, thinner, and more spindle-like when attached to plastic compared to unselected MSCs which spread wider along the surface of the culture flask, the so-called “fried egg” appearance. The doubling time of GD2-selected MSCs was 30 hrs compared to 90 hrs for unselected cells representing a 3-fold greater growth rate. Cell cycle analysis by flow cytometry showed ∼80% of cells were in G0/G1 and ∼20% were in S/G2/M phases of the cell cycle in both populations. With the shorter doubling time, this data indicates that GD2-selected MSCs move through the cell cycle more rapidly than unselected cells. In accordance with this finding, electron microscopy showed few organelles in the GD2-selected cells, but increase lamellar bodies indicating overall less complexity, but consistent with a greater membrane turnover rate (cell division) than unselected MSCs. Moreover, flow cytometric analysis revealed an increased expression of receptors for bFGF and EFG, known mitogenic factor receptors for MSCs, compared to unselected MSCs. In vitro differentiation of GD2-selected MSCs showed a more robust osteoid matrix formation (osteoblast) and proteoglycan formation (chondroblast) assayed by semi-quantitative Alizarin Red and Alcian blue staining, respectively. Additionally, more GD2-selected MSCs differentiated to adipocytes than among unselected cells. Surprisingly, GD2 expression persisted on the in vitro human MSC-differentiated osteoblasts, chondroblasts, and adipocytes, in contrast to human bone-derived osteoblasts, adipose tissue, and cartilage which lacked GD2 expression. We conclude that GD2 is a unique, stably expressed surface MSC marker which can be used to prospectively isolate MSCs from marrow, GD2-selcted cells have a more robust in vitro proliferation and differentiation potential which may be valuable for cell therapy, and biologically, in vitro isolated MSCs may not represent the in vivo progenitor for bone, fat, or cartilage.

scholarly journals Molecular Characterization of Bone Marrow and Peripheral Blood Compartments from Patients with Plasma Cell Leukemia in the Mmrf Commpass Study

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 1782-1782
Author(s):  
Sheri Skerget ◽  
Austin Christofferson ◽  
Sara Nasser ◽  
Christophe Legendre ◽  
The MMRF CoMMpass Network ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Multiple Myeloma ◽  
Bone Marrow ◽  
Flow Cytometry ◽  
Plasma Cell ◽  
Peripheral Blood ◽  
Research Funding ◽  
Plasma Cells ◽  
Cell Cycle Control ◽  
Cell Leukemia

Plasma cell leukemia (PCL) is rare but represents an aggressive, advanced form of multiple myeloma (MM) where neoplastic plasma cells (PCs) escape the bone marrow (BM) and circulate in the peripheral blood (PB). Traditionally, PCL is defined by the presence of >20% circulating plasma cells (CPCs), however, recent studies have suggested that PCL be redefined as the presence of >5% CPCs. The Multiple Myeloma Research Foundation CoMMpass study (NCT01454297) is a longitudinal, observational clinical study with 1143 newly diagnosed MM patients. BM-derived MM samples were characterized using whole genome (WGS), exome (WES), and RNA (RNAseq) sequencing at diagnosis and each progression event. When >5% CPCs were detected by flow cytometry, PCs were enriched independently from both compartments, and T-cells were selected from the PB as a control for WGS and WES. This substudy within CoMMpass provides the largest, most comprehensively characterized dataset of matched MM and PCL samples to date, which can be leveraged to better understand the molecular drivers of PCL. At diagnosis, 813/1143 CoMMpass patients had flow cytometry data reporting the percent PCs in PB, of which 790 had <5%, 17 had 5-20%, and 6 had >20% CPCs. Survival analyses revealed that patients with 5-20% CPCs (median = 20 months) had poor overall survival (OS) outcomes compared to patients with <5% CPCs (median = 74 months, p < 0.001), and no significant difference in outcome was observed between patients with 5-20% and >20% (median = 38 months) CPCs. Patients with 1-5% CPCs (median = 50 months, HR = 2.45, 95% CI = 1.64 - 3.69, p < 0.001) also exhibited poor OS outcomes compared to patients with <1% CPCs (median = 74 months), suggesting that patients with >1% CPCs are a higher risk population, even if they do not meet the PCL threshold. Using a cutoff of >5% CPCs, 23/813 (2.8%) patients presented with primary PCL (pPCL) at diagnosis. Of these patients, 7 (30%) were hyperdiploid (HRD), of whom 1 had a CCND1 and 1 had a MYC translocation; while 16 (70%) were nonhyperdiploid (NHRD), all of whom had a canonical immunoglobulin translocation (6 CCND1, 5 WHSC1, 3 MAF, 1 MAFA, and 1 MAFB). Of 124 patients with serial sample collections, 5 (4%) patients without pPCL had >5% CPCs at progression, and thus relapsed with secondary PCL (sPCL). Of the 5 sPCL patients, 2 (40%) were NHRD with a CCND1 or MAF translocation; while 3 (60%) were HRD, 1 with a WHSC1 translocation. Median time to diagnosis of sPCL was 22 months (range = 2 - 31 months), and patients with sPCL (median = 22 months) and pPCL (median = 30 months) exhibited poor OS outcomes as compared to MM patients (74 months, p < 0.001). Sequencing data was available for 15 pPCL and 5 sPCL samples. For 12 patients with WES, WGS, and RNAseq performed on their PCL tumor sample, an integrated analysis identified recurrent, complete loss-of-function (LOF) events in only CDKN2C/FAF1, SETD2, and TRAF3. Five pPCL patients had complete LOF of a gene involved in G1/S cell cycle control, including CDKN2C, CDKN2A, CDKN1C, and ATM. These LOF events were not observed in NHRD t(11;14) PCL patients, suggesting that CCND1 overexpression and LOF of genes involved in G1/S cell cycle control may represent independent drivers of PCL. Comparing WES and WGS data between matched MM and PCL tumor samples revealed a high degree of similarity in mutation and copy number profile. However, differential expression analysis performed for 13 patients with RNAseq data comparing their MM and PCL tumors revealed 27 up- and 39 downregulated genes (padj < 0.01, FDR = 0.1) in PCL versus MM. Pathway analysis revealed an enrichment (p < 0.001) for genes involved in adhesion and diapedesis, including upregulation of ITGB2, PF4, and PPBP, and downregulation of CCL8, CXCL12, MMP19, and VCAM1. The most significantly downregulated gene in PCL (log2FC = -6.98) was VCAM1, which plays a role in cell adhesion, and where loss of expression (TPM < 0.01) was observed across all PCL samples. Upregulation of four S100 genes including S100A8, S100A9, S100A12, and S100P, which have been implicated in tumor growth, metastasis, and immune evasion, was also observed in PCL. Interestingly, a S100A9 inhibitor has been developed and may represent a novel treatment option for PCL patients. In summary, PCL was found to be associated with molecular events dysregulating G1/S cell cycle control coupled with subtle changes in transcription that likely occur in a subclonal population of the MM tumor. Disclosures Lonial: Genentech: Consultancy; GSK: Consultancy; BMS: Consultancy; Janssen: Consultancy, Research Funding; Karyopharm: Consultancy; Takeda: Consultancy, Research Funding; Celgene Corporation: Consultancy, Research Funding; Amgen: Consultancy.

Deficiency of Rac1 and Rac2 GTPases Perturbs Erythroid Proliferation and Differentiation but Not Enucleation.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 467-467 ◽  
Author(s):  
Theodosia A. Kalfa ◽  
Suvarnamala Pushkaran ◽  
Jose A. Cancelas ◽  
Michael Jansen ◽  
James F. Johnson ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
Ex Vivo ◽  
Terminal Differentiation ◽  
Fluorescent Label ◽  
Spleen Cells ◽  
Hematopoietic Stem ◽  
Erythroid Precursor ◽  
Survival Difference ◽  
Erythroid Precursors

Abstract The small Rho GTPases Rac1 and Rac2 have overlapping as well as distinct roles in actin organization, cell survival, and proliferation in various hematopoietic cell lineages. However their role in erythropoiesis has not yet been fully elucidated. Using conditional gene-targeted mice we demonstrated that deficiency of Rac1 and Rac2 GTPases causes a significant phenotype in erythroid lineage. The mice develop anemia that is both hemolytic (abnormal structure of the erythrocyte cytoskeleton and decreased deformability; Kalfa et al. Blood 2006) and dyserythropoietic in nature. Cre-recombinase-induced deletion of Rac1 genomic sequence was accomplished as previously described (Gu et al. Science, 2003) on a Rac2-null genetic background. Colony assays revealed that although BFU-E frequency was similar, Rac1−/ −;Rac2−/ − BFU-E colonies had a strikingly different morphology appearing as round, small, dense colonies with solid edges, likely a manifestation of migration defects associated with Rac GTPase deficiency. CFU-E formation from hematopoietic stem/progenitors (HSC/Ps) derived from bone marrow (BM) of Rac1−/ −;Rac2−/ − mice was decreased more than 50% in comparison to WT (p=0.01). On the other hand, Rac1−/ −;Rac2−/ − mice developed marked splenomegaly (2-fold enlargement) and low density spleen cells demonstrated a 2-fold increase in CFU-E frequency in comparison to WT (p=0.008). To further assess erythroblast differentiation, BM and spleen cells were immunostained with fluorescent label-conjugated anti-CD71 and anti-Ter119, as previously described (Socolovski et al. Blood, 2001). Flow cytometry analysis revealed that the BM content of proerythroblasts and basophilic erythroblasts was significantly decreased (&gt;5-fold) in Rac1−/ −;Rac2−/ − vs. WT mice. In contrast, the same erythroblast populations were 4-fold increased in the spleens of Rac1−/ −;Rac2−/ − animals. However, the terminal differentiation to orthochromatic erythroblasts was comparable. No survival difference was found between WT and Rac1−/ −;Rac2−/ − erythroid precursors by flow cytometry with annexin-V, indicating that apoptosis was not contributing to the changes in erythropoiesis in Rac-deficient mice. Differentiation of Rac1−/ −;Rac2−/ − HSC/Ps to proerythroblasts and basophilic erythroblasts was delayed significantly at the early stages in ex vivo erythropoiesis culture (Giarratana et al. Nat Biotechnol, 2005) in the presence of SCF and erythropoietin. Later in the culture the cytokine-independent terminal differentiation to orthochromatic erythroblasts was similar between WT and Rac1−/ −;Rac2−/ − mice. The phosphorylation of AKT in WT and Rac1−/ −;Rac2−/ − erythroid precursors revealed by immunoblotting was similar, but the phosphorylation of extracellular signal-regulated kinase (ERK) (p42/p44) in Rac1−/ −;Rac2−/ − erythroid precursors was significantly decreased. The enucleation process was evaluated quantitatively, in ex vivo erythropoiesis cultures, by flow cytometry, using SYTO16, a cell-permeable DNA-staining dye. The frequency of enucleated red cells (SYTO16-negative, Ter119-positive population) was similar in the WT and Rac1−/ −;Rac2−/ − erythroid cultures. These data suggest that Rac1 and Rac2 deficiency does not affect enucleation but causes a significant decrease of early erythroid precursor populations in the bone marrow.

Donor Th17 and Th1 Contribute to Chronic Graft-Versus-Host Disease

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 727-727
Author(s):  
Hisakazu Nishimori ◽  
Haruko Sugiyama ◽  
Koichiro Kobayashi ◽  
Yoshiko Yamasuji ◽  
Sachiyo Kadohisa ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
T Cells ◽  
Th1 Cells ◽  
Flow Cytometry Analysis ◽  
Spleen Cells ◽  
Graft Versus Host ◽  
Donor T Cells ◽  
Ifn Γ ◽  
Deficient Mice

Abstract Abstract 727 Chronic graft-versus-host disease (cGVHD) remains a major cause of late death and morbidity after allogeneic hematopoietic cell transplantation, and the treatment of cGVHD remains challenging. All-trans retinoic acid (ATRA), a potent derivative of vitamin A, can regulate immune responses. Am80, which has biological activity approximately 10 times more potent than that of ATRA by binding to RARα and RARβ, but not RARγ, also reduces the severity and progression of immune disease models, including contact dermatitis, collagen-induced arthritis, allergic encephalomyelitis, and atherosclerosis models. Am80 has been reported to suppress the differentiation of Th17 and Th1 cells. Previously, we demonstrated that the administration of ATRA or Am80, significantly decreased skin fibrosis and alopecia, a dominant feature of cutaneous cGVHD, using a well-characterized experimental model of cGVHD: B10.D2 (H−2d) → BALB/c (H−2d). Flow cytometry analysis of the peripheral lymph nodes (PLNs) on day 16 showed significant reductions in Th1, Th17, and Foxp3+ regulatory T cells (Tregs) in Am80-treated recipients, as compared to controls, whereas no reduction in Th2 cells was observed (Nishimori et al., 2009 ASH). To further investigate the roles of Th17 cells and other Th subsets (Th1 and Th2) in cGVHD, we first assessed the kinetics of Th cytokines in the B10.D2 (H-2d) → BALB/c (H-2d) model of cGVHD, which reflects the clinical and pathological symptoms of human cGVHD. BALB/c mice were subjected to sublethal irradiation (6.75 Gy) and injected with 8 × 106 T-cell-depleted bone marrow cells and 8 × 106 spleen cells or 2 × 106 CD90+ spleen T cells from B10.D2 mice. In the early phase (day 14) of post bone marrow transplant (BMT), donor T cells produced significantly less IL-17 in the PLNs of recipients in allogeneic BMT than in syngeneic BMT (1.68±0.09% vs. 4.30±0.53%, p<0.01), while in the late phase (day 28) donor T cells that produced IL-17 were increased significantly in allogeneic recipients, compared with syngeneic controls (7.76±0.27% vs. 1.61±0.16%, p < 0.005). Both IL-17−/IFN-γ+ and IL-17/IFN-γ double positive cells (Th17/Th1 cells) were constantly detected more frequently in allogeneic recipients (p<0.05). Next, we evaluated whether Th17 contributes in a model utilizing IL-17-deficient mice on a B10.D2 background as donor mice; these mice were backcrossed for more than six generations from the original knock-out (KO) mice. Upon transfer of IL-17−/− B10.D2 donor T cells in allogeneic BMT models, skin cGVHD was significantly ameliorated, as compared to recipients of wild-type (WT) T cells, as shown in Figure (p=0.02). Histopathological examination of the skin showed significantly reduced cGVHD pathology in recipients of IL-17−/− donors (3.17±1.09 vs. 8.50±0.84, p=0.006). Flow cytometry analysis of the PLNs, mesenteric lymph nodes (MLNs), and spleen cells in the early phase (day 14) showed no differences in Th1, Th2, and Tregs, while recipients of IL-17−/− had significantly fewer Th1 (p<0.05) and Th17/Th1 (p<0.01) cells on day 35. Furthermore, IFN-γ deficient mice on a B10.D2 background were also backcrossed from the original KO mice and cGVHD was evaluated. BMT from IFN-γ−/− donors significantly improved the clinical cGVHD score, compared with WT donors (p<0.05). Allogeneic recipients given anti-IFN-γ mAb (500 μg/mouse) on days 0, 5, 10, and 15 post BMT had significantly less severe cGVHD, compared with control antibody recipients (p<0.05). Taken together, Th17, especially in the late-chronic phase, and Th1 contribute to the development of cGVHD by promoting the production of proinflammatory cytokines. Targeting Th17 and Th1 may be a promising strategy for preventing and treating cGVHD. Disclosures: No relevant conflicts of interest to declare.

Steady State Differences In Metabolic Properties Of Bone Marrow Versus Spleen Erythroid Cells

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 943-943 ◽  
Author(s):  
Genís Campreciós ◽  
Jeffrey Barminko ◽  
Jeffrey Bernitz ◽  
Saghi Ghaffari
Keyword(s):  
Cell Cycle ◽  
Bone Marrow ◽  
Flow Cytometry ◽  
S Phase ◽  
Erythroid Cells ◽  
Wild Type ◽  
Mitochondrial Mass ◽  
Stress Erythropoiesis ◽  
Erythroid Precursors ◽  
Metabolic Conditions

Abstract Erythropoiesis in adult mice occurs principally in the bone marrow, while stress erythropoiesis is mostly localized in the spleen. Although both bone marrow and spleen produce fully functional mature red blood cells, we noticed that the rate of maturation of erythroid precursors (based on the ratio of mature/immature erythroid cells) is over 10 times higher in the spleen as compared to the bone marrow in C57BL6 mice (3.7 ± 0.6 in the spleen as compared to 0.4 ± 0.1 in the bone marrow, n ≥ 10 mice). Cell cycle analysis of erythroid precursors revealed that bone marrow erythroid cells cycle up to 3 times more than their spleen counterparts (88 ± 1% vs 25 ± 7% of cells in the S phase, n=3). As reactive oxygen species (ROS) influence cell cycle, we measured ROS levels by flow cytometry using the CM-H2DCFDA probe. To our surprise, we found ROS levels to decrease (rather than increase) progressively in the bone marrow erythroid cells as they mature and accumulate hemoglobin. Interestingly, the levels of ROS were twice as high in the spleen erythroid cells as compared to erythroid cells in the bone marrow. As mitochondria are a major site of ROS production we measured mitochondrial mass by flow cytometry using Mitotracker Green. Mitochondrial mass was found to be two fold lower in the spleen erythroid cells as compared to the bone marrow. In agreement with these findings, qRT-PCR expression analysis of different antioxidant enzymes such as gluthathione peroxidases Gpx1 and Gpx4 showed higher levels in the bone marrow as compared to the spleen erythroid precursors. In particular, Gpx1 expression increased ten fold during erythroid maturation in the bone marrow while in the spleen the expression of Gpx1 did not change significantly. Together these results suggest erythroid metabolic profile is distinct in the spleen as compared to the bone marrow at the steady state. In order to compare homeostatic versus stress erythropoiesis we analyzed bone marrow and spleen from Foxo3-/- and Th3/+ thalassemic mice, two models of ineffective erythropoiesis with different degrees of severity and splenomegaly. As anticipated Foxo3-/- and Th3/+ erythroid precursors displayed decreased rate of maturation as compared to wild type cells in both bone marrow (0.3 ± 0.02 and 0.12 ± 0.01 for Foxo3-/- and Th3/+ respectively, n ≥ 10) and spleen (2.1 ± 0.3 and 0.42 ± 0.1 for Foxo3-/- and Th3/+ respectively, n ≥ 10 mice per group) and cell cycle analysis showed an increased number of cells in the S phase in both Foxo3-/- and Th3/+ spleen erythroid cells as compared to wild type (63 ± 2% and 79 ± 2% respectively, n=3). Unexpectedly however, ROS levels in both Foxo3-/- and Th3/+ spleen erythroid cells were decreased as compared to their wild type counterparts. The observed inverse correlation between cell cycling and ROS levels was further supported by expression analysis of Gpx1 and Gpx4, the levels of which were increased in Foxo3-/- as compared to wild type spleen erythroid cells. Collectively our results highlight the different erythroid metabolic conditions in the bone marrow versus spleen under both homeostatic and disease states. Further investigations elucidating differences in metabolic conditions and properties of erythroid cells in bone marrow versus spleen should improve our understanding of generation of erythroid cells under stress and the production of erythroid cells in vitro. Disclosures: No relevant conflicts of interest to declare.

A Novel Camptothecin Derivative 3j Inhibits Nsclc Proliferation Via Induction of Cell Cycle Arrest By Topo I-Mediated DNA Damage

2019 ◽  
Vol 19 (3) ◽  
pp. 365-374 ◽  
Author(s):  
Yang Liu ◽  
Jingyin Zhang ◽  
Shuyun Feng ◽  
Tingli Zhao ◽  
Zhengzheng Li ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Flow Cytometry ◽  
Dna Damage ◽  
Cyclin D ◽  
Dna Relaxation ◽  
Cycle Arrest ◽  
H460 Cell ◽  
H1975 Cell

Objective: The aim of this study is to investigate the inhibitory effect of camptothecin derivative 3j on Non-Small Cell Lung Cancer (NSCLCs) cells and the potential anti-tumor mechanisms. Background: Camptothecin compounds are considered as the third largest natural drugs which are widely investigated in the world and they suffered restriction because of serious toxicity, such as hemorrhagic cystitis and bone marrow suppression. Methods: Using cell proliferation assay and S180 tumor mice model, a series of 20(S)-O-substituted benzoyl 7- ethylcamptothecin compounds were screened and evaluated the antitumor activities in vitro and in vivo. Camptothecin derivative 3j was selected for further study using flow cytometry in NSCLCs cells. Cell cycle related protein cyclin A2, CDK2, cyclin D and cyclin E were detected by Western Blot. Then, computer molecular docking was used to confirm the interaction between 3j and Topo I. Also, DNA relaxation assay and alkaline comet assay were used to investigate the mechanism of 3j on DNA damage. Results: Our results demonstrated that camptothecin derivative 3j showed a greater antitumor effect in eleven 20(S)-O-substituted benzoyl 7-ethylcamptothecin compounds in vitro and in vivo. The IC50 of 3j was 1.54± 0.41 µM lower than irinotecan with an IC50 of 13.86±0.80 µM in NCI-H460 cell, which was reduced by 8 fold. In NCI-H1975 cell, the IC50 of 3j was 1.87±0.23 µM lower than irinotecan (IC50±SD, 5.35±0.38 µM), dropped by 1.8 fold. Flow cytometry analysis revealed that 3j induced significant accumulation in a dose-dependent manner. After 24h of 3j (10 µM) treatment, the percentage of NCI-H460 cell in S-phase significantly increased (to 93.54 ± 4.4%) compared with control cells (31.67 ± 3.4%). Similarly, the percentage of NCI-H1975 cell in Sphase significantly increased (to 83.99 ± 2.4%) compared with control cells (34.45 ± 3.9%) after treatment with 10µM of 3j. Moreover, increased levels of cyclin A2, CDK2, and decreased levels of cyclin D, cyclin E further confirmed that cell cycle arrest was induced by 3j. Furthermore, molecular docking studies suggested that 3j interacted with Topo I-DNA and DNA-relaxation assay simultaneously confirmed that 3j suppressed the activity of Topo I. Research on the mechanism showed that 3j exhibited anti-tumour activity via activating the DNA damage response pathway and suppressing the repair pathway in NSCLC cells. Conclusion: Novel camptothecin derivative 3j has been demonstrated as a promising antitumor agent and remains to be assessed in further studies.

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