Bone Marrow Stromal Cells Protect Both FLT3-ITD and FLT3-WT Primary AML Cells from the Anti-Leukemic Activity of the FLT3 Inhibitor AC220

Abstract FTL3 mutations are found in about 30% of AML patients, conferring a leukemic blast growth advantage, drug therapy resistance in the bone marrow (BM) and poor outcome. Mesenchymal stem/stromal cells (MSCs) are essential components of the bone marrow microenvironment, and growing evidence suggest that MSCs play a critical role in AML chemo-resistance, although the molecular mechanisms involved are poorly understood. The purpose of the study was to (1) establish an novel in vitro co-culture system between primary AML blasts and healthy donor BM-MSCs (HD-MSCs) or AML patient-derived MSCs (AML-MSCs), (2) evaluate the impact of culture with BM-MSCs on the sensitivity of AML cells to AC220 using patients samples with FLT3-ITD (n=4) or FLT3-WT (n=3). We first cultured HD-MSCs (n=5) and AML-MSC (n=3) and observed no phenotypical differences (CD14- CD34- CD45- CD73+ CD90+ CD105+), although HD-MSCs grew faster. We evaluated the effect of co-culturing AML samples (n=6) with HD-MSCs or AML-MSCs for 5 and 12 days on leukemic cell growth and found that both types of MSCs significantly and equally enhanced AML cell proliferation while maintaining blast phenotype. Using clonogenic assays on 4 AML specimens cultured alone or with either HD- or AML-MSCs for 5 and 12 days, we found that co-culture with either source of BM-MSCs drastically increased colony-forming cells number at day 5 and day 12 while CFC number decreased in the absence on BM-MSCs (no colonies at day 12 for the 4 samples), indicating that AML co-culture with HD/AML-MSCs supports the survival and/or proliferation of AML stem/progenitor cells. We next assessed the effect of increasing doses of AC220 (1, 10, 50, 100 and 500nM) on the apoptosis of FLT3-ITD (n=3) and FLT3-WT (n=4) AML cells cultured alone or with HD-MSCs. Exposure to AC220 for 72 hours significantly, and in a dose-dependent manner, increased the apoptosis of AML FLT3-ITD cells in monoculture (n=3, 21±1% of Annexin V positive cells for control, AC220 1nM 29±3.7%, 10nM 31±2.5%, 50nM 32±1.5%, 100nM 34±1.7% and 500nM 38±3.6%). In contrast, AML FLT3-ITD cells co-cultured with HD-MSCs were resistant to the drug (n=3, 21±2.6% of Annexin V positive cells for control, AC220 1nM 23±3%, 10nM 22±3%, 50nM 25±5.7%, 100nM 30±8.3% and 500nM 33±9.5%). Interestingly, we found that AML FLT3-WT are much less sensitive to increasing doses of AC220 compared to ITD samples (n=4, 27±3.9% of Annexin V positive cells for control, AC220 1nM 30±6.5%, 10nM 35±14%, 50nM 37±11%, 100nM 39±13% and 500nM 43±11%), and co-culture with BM-MSCs further decreased the sensitivity of AML FLT3-WT cells to AC220-induced apoptosis (n=4, 19±3.2% of Annexin V positive cells for control, AC220 1nM 17±3.9%, 10nM 20±3.4%, 50nM 19±3.7%, 100nM 21±4.5% and 500nM 26±1%). AC220 treatment for 3 days significantly, and in a dose-dependent manner, inhibited CFCs in AML FLT3-ITD (n=4, with 26±8%, 46±6%, 60±9%, 69±10% and 86±3% inhibition with 1, 10, 50, 100 and 500nM of AC220 respectively) while AML FLT3-ITD co-culture with HD-MSCs were less sensitive (n=4, with 9±10%, 30±6%, 42±9%, 57±11% and 72±7% inhibition with 1, 10, 50, 100 and 500nM of AC220, respectively). Similarly to the AC220-induced apoptosis, we observed that AML FLT3-WT CFCs are less sensitive to AC220-induced growth inhibition compared to ITD samples, although a 3 days exposure to AC220 significantly, and in a dose-dependent manner, inhibited AML FLT3-WT CFCs (n=3, with 38±16%, 44±14%, 58±12%, 70±21% and 81±19% inhibition with 1, 10, 50, 100 and 500nM of AC220, respectively). Interestingly, we observed that co-culture of AML FLT3-WT with stromal cells were significantly more resistant to increasing doses of AC220 (n=3, with 22±7%, 36±5%, 43±8%, 46±8% and 57±6% inhibition with 1, 10, 50, 100 and 500nM of AC220, respectively). Altogether, these results suggest that AML FLT3-ITD cells in monoculture are more sensitive to AC220 treatment compared to AML FLT3-WT primary cells, but more importantly, upon interaction with primary HD-MSCs, both WT and FLT3-ITD primary samples are protected from apoptosis and growth inhibition induced by AC220, indicating a critical role for the BM microenvironment in AC220 resistance. We are currently testing the impact of BM-MSCs co-culture on leukemic stem cell sensitivity to AC220 using transplantation in NSG mice. We will also evaluate if this co-culture model can be predictive of the response to in vivo treatment with AC220 in a patient-derived xenograft model. Disclosures Dos Santos: Janssen R&D: Research Funding. Danet-Desnoyers:Janssen R&D: Research Funding.

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Disruption of Leukemia/Stroma Cell Interactions by CXCR4 Antagonist CTCE-9908 Enhances Chemotherapy-Induced Apoptosis in AML

Blood ◽

10.1182/blood.v112.11.2415.2415 ◽

2008 ◽

Vol 112 (11) ◽

pp. 2415-2415

Author(s):

Hongbo Lu ◽

Zhihong Zeng ◽

Yuexi Shi ◽

Sergej Konoplev ◽

Donald Wong ◽

...

Keyword(s):

Bone Marrow ◽

Stromal Cells ◽

Leukemia Cell ◽

Bone Marrow Microenvironment ◽

Cell Interactions ◽

Leukemic Cells ◽

Dependent Manner ◽

Induced Apoptosis ◽

Dose Dependent

Abstract The chemokine receptor CXCR4 is critically involved in the migration of hematopoietic cells towards the stromal derived factor (SDF-1α)-producing bone marrow microenvironment. We and others have previously demonstrated that stroma/leukemia interactions mediate protection of leukemic cells from chemotherapy-induced apoptosis (Konopleva, Leukemia 2002). Using a peptide analog of SDF-1α designated CTCE-9908, we tested the hypothesis that CXCR4 inhibition interferes with stromal/leukemia cell interactions resulting in increased sensitivity to chemotherapy. Our results showed that CTCE-9908 significantly inhibits SDF-1α-induced migration of U937 (43% inhibition) and OCI-AML3 cells (40% inhibition) in a dose-dependent manner. In three of the four primary AML samples which expressed CXCR4 on cell surface and migrated in response to SDF-1α, 50 μg/ml CTCE-9908 reduced SDF-1α-induced migration of leukemic blasts (60%, 19% and 50% inhibition respectively). In in vitro co-culture systems, stromal cells significantly protected OCI-AML3 cells from chemotherapy induced apoptosis [no MS-5, 75.2±5.2% annexinV(+); with MS-5, 59±1.1% annexinV(+)]. Western blot analysis revealed that CTCE-9908 inhibits Akt and Erk phosphorylation in a dose-dependent manner in the OCI-AML3 cell line stimulated by SDF-1α. Blockade of CXCR4 expression with CTCE-9908 markedly abrogated the protective effects of stromal cells on OCI-AML3 [Ara-C, 59±1.1% annexinV(+); Ara-C + CTCE-9908, 76.9±1.35 annexinV(+)]. Most importantly, it decreased stroma-mediated protection from AraC-induced apoptosis in four out of five primary AML samples with surface expression of functional CXCR4 (mean increase, 25.1±9.3% compared to chemotherapy alone). In vivo, subcutaneous administration of 1.25mg CTCE-9908 induced mobilization of leukemic cells from primary AML patient transplanted into NOD/Scid-IL2Rγ-KO mice (from 15% to 27% circulating leukemic cells 1 hour post CTCE-9908 injection). Taken together, our data suggest that SDF-1α/CXCR4 interactions contribute to the resistance of leukemic cells to chemotherapy-induced apoptosis via retention of leukemic cells in the bone marrow microenvironment niches. Disruption of these interactions by the potent CXCR4 inhibitor CTCE-9908 represents a novel strategy for targeting leukemia cell/bone marrow microenvironment interaction. Based on these observations, in vivo experiments are ongoing to characterize the efficacy of chemotherapy combined with CTCE-9908.

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Activity of Targeted Molecular Therapeutics Against Primary AML Cells: Putative Role of the Bone Marrow Microenvironment.

Blood ◽

10.1182/blood.v106.11.2304.2304 ◽

2005 ◽

Vol 106 (11) ◽

pp. 2304-2304 ◽

Cited By ~ 1

Author(s):

Teresa McQueen ◽

Marina Konopleva ◽

Michael Andreeff

Keyword(s):

Bone Marrow ◽

Stromal Cells ◽

Culture System ◽

Hematological Malignancies ◽

Annexin V ◽

Leukemic Cells ◽

Mek Inhibitors ◽

Molecular Therapeutics ◽

Induced Apoptosis

Abstract In hematological malignancies, there are reciprocal interactions between leukemic cells and cells of the bone marrow (BM) microenvironment such as mesenchymal stem cells (MSC). It is speculated that specific BM niches may provide a sanctuary for subpopulations of leukemic cells to evade chemotherapy-induced death and allow acquisition of a drug-resistant phenotype. In this study, we compared anti-leukemia effects of Ara-C and various signal transduction and apoptosis inhibitors in a co-culture system of primary AML and human bone marrow-derived MSC. AML blasts from 11 primary AML samples with high (>70%) blast count were co-cultured with MSC for 24 hours, after which they were exposed to the indicated concentrations of inhibitors for 48–96 hrs. Concentrations were selected on the basis of preliminary cell line studies which determined efficient inhibition of drug targets. Induction of apoptosis was analyzed by Annexin V flow cytometry after gating on the CD90 APC(−) (non-MSC) population. MSC protected leukemic blasts from spontaneous apoptosis in all 11 samples studied (mean annexinV positivity, 49.5±7.2% vs 25.3±4.8%, p<0.001) and from Ara-C-induced cytotoxicity in 6 out of 11 samples (p=0.02). No difference in the degree of protection was noted when MSC from older vs. younger donors were used (not shown). Co-culture of leukemic cells with MSC resulted in significant (p<0.03) suppression of inhibitor-induced apoptosis for all agents tested (Table 1), however PI3K/AKT inhibitors seemed to overcome MSC-mediated resistance. In addition, specific inhibitors of Bcl-2 and MDM2 induced apoptosis not only in suspension, but also in the MSC co-culture system, while Raf-1/MEK inhibitors were less effective. The AKT inhibitor A443654 caused apoptosis induction not only in leukemic cells, but also in MSC, which likely accounted for its high efficacy in stromal co-cultures (53±6% annexin V+). In a different study (Tabe et al, ASH 2005), we report that interactions of leukemic and BM stromal cells result in the activation of PI3K/ILK/AKT signaling in both, leukemic and stromal cells. We therefore propose that disruption of these interactions by specific PI3K/AKT inhibitors represents a novel therapeutic approach to eradicate leukemia in the BM microenvironment via direct effects on leukemic cells and by targeting activated BM stromal cells. Furthermore, Bcl-2 and MDM2 inhibitors appear to retain their efficacy in stroma-cocultured AML cells, while the efficacy of chemotherapy and Raf/MEK inhibitors in these conditions may be reduced. Further studies are aimed at the elucidation of the role of the BM microenvironment and its ability to activate specific signaling pathways in the pathogenesis of leukemias and on efforts to disrupt the MSC/leukemia interaction (Zeng et al, ASH 2005). Focus on this stroma-leukemia-stroma crosstalk may result in the development of strategies that enhance the efficacy of therapies in hematological malignancies and prevent the acquisition of a chemoresistant phenotype. Table 1. Leukemia Cell Apoptosis in a MSC/AML Co-Culture System Target Bcl-2/XL MDM2 PI3K AKT Raf-1 MEK Apoptosis was determined as percentage of Annexin V(+)CD90(−) cells, and calculated by the formula: % specific apoptosis = (test − control) x 100 / (100 − control). Compound, concentration Ara-C, 1 μM ABT-737, 0.1 μM Nutlin-3A, 2.5 μM LY294002, 10 μM A443654, 1 μM BAY43-9006, 2.5 μM CI1040, 3 μM AML 28±7 69±7 45±7 53.8±13.3 75±7 35±11 27±11 AML + MSC 16±4 38±6 28±6 31.2±6.9 53±6 18±8 15±5

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The New CXCR4 Inhibitor MDX-1338 Exerts Anti-Tumor Activity in Multiple Myeloma

Blood ◽

10.1182/blood.v118.21.1844.1844 ◽

2011 ◽

Vol 118 (21) ◽

pp. 1844-1844 ◽

Cited By ~ 1

Author(s):

Aldo M Roccaro ◽

Antonio Sacco ◽

Michelle Kuhne ◽

AbdelKareem Azab ◽

Patricia Maiso ◽

...

Keyword(s):

Multiple Myeloma ◽

Bone Marrow ◽

Tumor Cells ◽

Mouse Models ◽

Research Funding ◽

Therapeutic Agents ◽

Dependent Manner ◽

Apoptotic Pathways ◽

Dose Dependent

Abstract Abstract 1844 Background. We have previously shown the SDF1/CXCR4 axis plays a major role in homing and trafficking of multiple myeloma (MM) to the bone marrow (BM), and disruption of the interaction of tumor cells with the BM leads to enhanced sensitivity to therapeutic agents. We hypothesize that the novel anti-CXCR4 antibody, BMS936564/MDX-1338, may prevent the homing and adhesion of MM cells to the BM and will sensitize them to therapeutic agents. Methods. Primary MM cells (CD138+); MM cell lines (MM.1S, RPMI.8226); and primary MM bone marrow stromal cells (BMSCs) were used. Migration towards SDF-1 and BMSCs has been evaluated. Cytotoxicity and DNA synthesis were measured by MTT and thymidine uptake, respectively. Cell signaling and apoptotic pathways were studied by Western Blot. Synergism was calculated using the Chou-Talalay method. In vivo MM tumor growth was evaluated with xenograft mouse models. Results. MDX-1338 inhibited migration of MM cells toward SDF-1a and primary MM BMSCs, in a dose-dependent manner. Adhesion of primary MM cells to BMSCs was also inhibited by BMS936564/MDX-1338 in a dose-dependent manner, while also inducing cytotoxicity on primary BM-derived CD138+ cells. BMS936564/MDX-1338 targeted MM cells in the context of BM milieu by overcoming BMSC-induced proliferation of tumor cells. In addition, BMS936564/MDX-1338 synergistically enhanced bortezomib-induced cytotoxicity in MM cells. BMS936564/MDX-1338-dependent activation of apoptotic pathways in MM cells was documented, as shown by cleavage of caspase-9 and PARP. SDF-1a-induced ERK-, Akt-, and Src-phosphorilation was inhibited by BMS936564/MDX-1338 in a dose-dependent manner. Importantly, BMS936564/MDX-1338 inhibited MM cell proliferation in vivo in xenograft mouse models. Conclusion. These studies therefore show that targeting CXCR-4 in MM by using BMS936564/MDX-1338 represents a valid therapeutic strategy in this disease. Disclosures: Roccaro: Roche:. Kuhne:BMS: Employment. Pan:Bristol-Myers Squibb: Employment. Cardarelli:Bristol-Myers Squibb: Employment. Ghobrial:Noxxon: Research Funding; Bristol-Myers Squibb: Research Funding; Millennium: Research Funding; Noxxon:; Millennium:; Celegene:; Novartis:.

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Inhibition Of PI3K Classia Kinases Using GDC0941 Overcomes Protection Of Multiple Myeloma Cells In The Bone Marrow Microenvironment

Blood ◽

10.1182/blood.v122.21.3169.3169 ◽

2013 ◽

Vol 122 (21) ◽

pp. 3169-3169

Author(s):

Hugh Kikuchi ◽

Amofa Eunice ◽

Maeve McEnery ◽

Farzin Farzaneh ◽

Stephen A Schey ◽

...

Keyword(s):

Multiple Myeloma ◽

Bone Marrow ◽

Cell Proliferation ◽

Bone Resorption ◽

Cell Lines ◽

Stromal Cells ◽

Conflicts Of Interest ◽

Pi3k Pathway ◽

Dependent Manner ◽

Dose Dependent

Abstract Despite of newly developed and more efficacious therapies, multiple myeloma (MM) remains incurable as most patient will eventually relapse and become refractory. The bone marrow (BM) microenvironment provides niches that are advantageous for drug resistance. Effective therapies against MM should ideally target the various protective BM niches that promote MM cell survival and relapse. In addition to stromal mesenchymal/myofibroblastic cells, osteoclasts play a key supportive role in MM cell viability. Additionally, 80% of patients develop osteolytic lesions, which is a major cause of morbidity. Increased osteoclast activity is characteristic in these patients and targeting osteoclast function is desirable to improve therapies against MM. Osteoclasts need to form an F-actin containing ring along the cell margin that defines a resorbing compartment where protons and degradative enzymes are secreted for dissolution of bone mineral. Remodelling of F-actin and vesicle secretion are regulated by the class IA PI3K pathway during osteoclastic bone resorption. Additionally, it has recently been shown that inhibition of the class IA PI3K pathway in MM cells with GDC0941 induces apoptosis-mediated killing. We hypothesised that GDC0941 could be used as a therapeutic agent to overcome MM-induced osteoclast activation. GDC0941 inhibited maturation of osteoclasts derived from BM aspirates from MM patients in a dose dependent manner. This correlated with decreased bone resorption of osteoclasts cultured on dentine discs. Exposure of mature osteoclasts to GC0941 resulted in abnormal organisation of larger F-actin rings, suggesting a negative effect on the dynamics of the actin cytoskeleton required for bone resorption. We also found that GDC-0941 can prevent protection of the MM cell lines MM1.S and MM1.R by osteoclasts against killing. GDC-0941 alone blocked MM cell proliferation independently of the presence of BM stromal cells and synergised with other therapeutic agents including Lenalidomide, Pomalidomide, Bortezomid and Dexamethasone. We also found that in the presence of MM cells, Dexamethasone (a drug commonly used alone or in combination with new drugs against MM) induced the proliferation of BM stromal cells and adhesion of MM cells on this protective stroma in a dose dependent manner. Dexamethasone is highly effective at MM cell killing when cells are cultured alone. However, we found that at low doses (below 1 uM) and in the presence of BM stromal cells, Dexamethasone could induce MM cell proliferation. GDC0941 enhanced Dexamethasone killing even in the presence of BM stromal cells by blocking Dexamethasone-induced stromal cell proliferation and adhesion of MM cells on the stroma. Targeting individual the PI3K Class IA isoforms alpha, beta, delta or gamma proved to be a less efficient strategy to enhance Dexamethasone killing. Previous work has shown that efficacy of targeting individual PI3K Class I A isoforms would be low for activation of caspases in MM cells as it would be dependent on relative amounts of isoforms expressed by the MM patient. GDC-0941 also inhibited the proliferation of MM1.R and RPMI8266 MM cell lines, which are less sensitive to treatment to Dexamethasone. Co-culture of MM cells with BM stromal cells induced the secretion of IL-10, IL-6, IL-8, MCP-1 and MIP1-alpha. The dose-dependant increased proliferation of Dexamethasone-treated MM cells in the presence of the BM stroma correlated with the pattern of secretion of IL-10 (a cytokine that can induce B-cell proliferation) and this was blocked by the combination of Dexamethasone with GDC0941. GDC-0941 alone or in combination with Dexamethasone was more efficacious at inducing MM cell apoptosis in the presence of the BM stroma cells vs treatment of MM cells alone. These are very encouraging results as they suggest that GDC-0941 in combination with Dexamethasone would be potentially highly efficacious for targeting MM cells in the BM microenvironment. We are currently performing in vivo data using C57BL/KaLwRij mice injected with 5T33-eGFP MM cells that will be discussed at the meeting. We propose that MM patients with active bony disease may benefit from treatment with GDC0941 alone or in combination with currently used therapeutic drugs against MM. Disclosures: No relevant conflicts of interest to declare.

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CXCR4 Inhibition as a Therapeutic Strategy in Leukemia.

Blood ◽

10.1182/blood.v104.11.456.456 ◽

2004 ◽

Vol 104 (11) ◽

pp. 456-456

Author(s):

Zhihong Zeng ◽

Randall L. Evans ◽

Ziwei Huang ◽

Michael Andreeff ◽

Marina Konopleva

Keyword(s):

Bone Marrow ◽

Flow Cytometry ◽

Stromal Cells ◽

Surface Expression ◽

Bone Marrow Microenvironment ◽

Jurkat Cells ◽

Leukemic Cells ◽

Dependent Manner ◽

Viable Cells ◽

Induced Apoptosis

Abstract The chemokine receptor CXCR4 is critically involved in the migration of hematopoietic cells to the stroma derived factor (SDF-1α)-producing bone marrow microenvironment. We and others have previously demonstrated that stromal-leukemic interactions mediate protection of leukemic cells from chemotherapy-induced apoptosis. (Konopleva et al, Leukemia 2002; Tabe, Konopleva, et al, Blood 2004; Burger JA et al., Blood 2000). Using peptide based CXCR4 inhibitors, derived from the chemokine viral macrophage inflammatory protein II (vMIP II), we tested the hypothesis that CXCR4 inhibition interferes with stromal/leukemia cell interactions resulting in increased sensitivity to chemotherapy. CXCR4 was highly expressed on the cell surface of CML myeloid blood crisis cells (KBM5), KBM5/STI-resistant cells, lymphoid CEM and Jurkat cells, myeloid leukemic OCI-AML3 and U937 cells. In contrast, NB4 and TF-1 cells expressed low-levels surface CXCR4, and no surface expression was detected on KG-1 and HL-60 leukemic cells. Among CXCR4(+) cell lines, Jurkat cells demonstrated the highest chemoattractive response to SDF-1α(23 +/− 0.03% migration at SDF-1α50ng/ml, and 54 +/− 0.01% at 100ng/ml). The ability of three CXCR4 inhibitors to inhibit chemotaxis of Jurkat cells was examined in a standard migration assay. Results indicate that D10-vMIP-II, a polypeptide with the first 10 amino acids substituted by the D isoform, exhibits the strongest antagonistic effect on SDF-1α or stromal cell induced chemoattraction. D10-vMIP-II also decreases CXCR4 surface expression in a concentration-dependent manner: flow cytometry and live cell confocal microscopy revealed that within 30min of exposure D10-vMIP-II causes CXCR4 internalization that persisted for at least 4 hrs at 0.01μM and for 24 hrs at 0.1μM. Analysis of SDF-1α-mediated signaling demonstrated that D10-vMIP-II inhibits AKT and ERK phosphorylation. Finally, we examined the effects of D10-vMIP-II on the response to chemotherapy of leukemic cells co-cultured with MS5 stromal cells. Pre-treatment of Jurkat cells enhanced doxorubicin-induced apoptosis: Doxorubicin alone (10μM) 75 +/− 0.07% viable cells compared to control; Doxorubicin and D10-vMIP-II: 53 +/− 0.04% viable cells. Furthermore, D10-vMIP-II enhanced the sensitivity of primary CLL cells to Fludarabine in the in vitro stromal co-culture system. CLL samples with high surface expression of CXCR4 (n=3) co-cultured with stromal MS-5 cells were pre-treated with 0.1μM D10-vMIP II followed by 10μM Fludarabine (9-β-D-arabinofuranosyl-2-fluoroadenine). Stromal cells prevented Fludarabine-induced killing (64%±16.2 viable cells in stromal co-culture compared to 31% viable cells in medium only). Inhibition of CXCR4 signaling abrogated this protective effect and diminished CLL cell survival (26.9±7.1% viable cells, p=0.03 compared to Fludarabine-treated CLL cells co-cultured with MS-5). This growth inhibition was mediated by apoptosis induction as determined by CD45/annexinV flow cytometry (DMSO, 14.49±5.3% annexinV(+) leukemic cells; Fludarabine, 47.2±24.9%; D10-vMIP II followed by Fludarabine, 61.3±18.9%). Taken together, our data suggest that SDF-1α/CXCR4 interactions contribute to the resistance of leukemic cells to chemotherapy-induced apoptosis. Disruption of these interactions by the potent CXCR4 inhibitor D10-vMIP-II represents a novel strategy for the targeting leukemic cells within their bone marrow microenvironment.

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The role of platelet α-granular proteins in the regulation of thrombopoietin messenger RNA expression in human bone marrow stromal cells

Blood ◽

10.1182/blood.v95.10.3094.009k05_3094_3101 ◽

2000 ◽

Vol 95 (10) ◽

pp. 3094-3101 ◽

Cited By ~ 5

Author(s):

Ranita Sungaran ◽

Orin T. Chisholm ◽

Boban Markovic ◽

Levon M. Khachigian ◽

Yoshihiro Tanaka ◽

...

Keyword(s):

Bone Marrow ◽

Growth Factor ◽

Mrna Expression ◽

Stromal Cells ◽

Messenger Rna ◽

Human Bone ◽

Human Bone Marrow ◽

Dependent Manner ◽

Platelet Factor ◽

Dose Dependent

Thrombopoietin (TPO), the specific cytokine that regulates platelet production, is expressed in human bone marrow (BM), kidney, and liver. There appears to be no regulation of TPO in the kidney and liver, but TPO messenger RNA (mRNA) expression can be modulated in the stromal cells of the BM. In this study, we used primary human BM stromal cells as a model to study the regulation of TPO mRNA expression in response to various platelet -granular proteins. We showed that platelet-derived growth factor (PDGF) BB and fibroblast growth factor (FGF) 2 stimulated TPO mRNA expression in both a dose-dependent and time-dependent manner. The addition of 50 ng/mL of PDGF and 20 ng/mL of FGF resulted in maximal induction of TPO mRNA expression in 4 hours. We also found that platelet factor 4 (PF4), thrombospondin (TSP), and transforming growth factor-beta (TGF-β) are negative modulators of megakaryocytopoiesis. We observed suppression in TPO mRNA expression with 1 μg/mL of both PF4 and TSP and 50 ng/mL of TGF-β, with maximal suppression occurring 4 hours after the addition of these proteins. Finally, the addition of whole-platelet lysate produced a dose-dependent inhibition of TPO expression. On the basis of these findings, we propose that the platelet -granular proteins studied may regulate TPO gene expression in BM stromal cells by means of a feedback mechanism.

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Bone marrow-derived mesenchymal stromal cells improve vascular regeneration and reduce leukocyte-endothelium activation in critical ischemic murine skin in a dose-dependent manner

Cytotherapy ◽

10.1016/j.jcyt.2014.05.008 ◽

2014 ◽

Vol 16 (10) ◽

pp. 1345-1360 ◽

Cited By ~ 16

Author(s):

Riccardo Schweizer ◽

Pranitha Kamat ◽

Dennis Schweizer ◽

Cyrill Dennler ◽

Shengye Zhang ◽

...

Keyword(s):

Bone Marrow ◽

Mesenchymal Stromal Cells ◽

Stromal Cells ◽

Dependent Manner ◽

Vascular Regeneration ◽

Dose Dependent

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Human Cytomegalovirus Inhibits Granulopoiesis Via Direct Infection and Inducing Apoptosis.

Blood ◽

10.1182/blood.v106.11.3859.3859 ◽

2005 ◽

Vol 106 (11) ◽

pp. 3859-3859

Author(s):

Qing Wen Wang ◽

Zheng Xian He ◽

Xiao Feng Li ◽

Wei Xiong ◽

Mei Lian Chen ◽

...

Keyword(s):

Bone Marrow ◽

Viral Infection ◽

Human Cytomegalovirus ◽

Hcmv Infection ◽

Annexin V ◽

Pcr Analysis ◽

Control Group ◽

Dependent Manner ◽

Ad169 Strain ◽

Dose Dependent

Abstract Human cytomegalovirus (HCMV) infection can cause delayed leukocyte recovery after bone marrow transplantation and often associated with suppression of granulocyte/macrophage progenitor. Leukocyte lineage may be one of the major sites of HCMV infection. However, whether HCMV can interfere with CFU-GM formation and induce apoptosis in granulocyte/macrophage progenitor have not been well investigated. Human bone marrow mononuclear cells (Ficoll), promyelocyte cell line HL-60 and HCMV AD169 strain were co-cultured. Each bone marrow specimen was HCMV DNA and HCMV IgM negative by PCR and ELISA test. Our results showed that HCMV significantly inhibited the formation of CFU-GM as shown in two different concentrations of viral infection groups: 139.26 ± 5.42 (2×105pfu/ml), and 124.19 ± 8.82 (2×106pfu/ml) (colonies/2×105cells/ml, n=26). These were significant differences compared with the blank control group (P<0.01, P<0.05) respectively. HCMV also significantly inhibited the growth of HL-60 cells in three different concentrations of viral infection groups (101pfu/ml, 102pfu/ml, 103pfu/ml). After incubation for 7 days, viability cells in control group and each infection groups (n=20) were 96%, 83%, 73%, and 64%, showing that HCMV infection decreased the viability of HL-60 cells in a dose-dependent manner. The apoptotic effect was also investigated by Annexin V assay using flow cytometry. The percentage of Annexin V -positive cells were 19.60 ± 1.63 (in group of 101pfu/ml) (P<0.05), 28.70 ± 2.61 (102pfu/ml) (P<0.05), and 36.3 ± 2.57 (103pfu/ml) (P<0.01), compared with control group 3.96 ± 1.75 (n=8). The apoptotic cells were further confirmed by morphologic observation and DNA ladder formation. Furthermore, HCMV DNA was positive in HL-60 cells and cells of CFU-GM measured by PCR and IS-PCR analysis respectively. These were negative in blank and mock control groups. Our data demonstrated that: (1) HCMV AD169 strain inhibited the growth of HL-60 and CFU-GM formation in a dose-dependent manner; (2) HCMV transcription occurred in HL-60 and CFU-GM cells; and (3) This virus induced apoptosis in HL-60 cells. HCMV may have an inhibiting effect on granulopoiesis via direct infection and inducing apoptosis on myeloid progenitors.

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The role of platelet α-granular proteins in the regulation of thrombopoietin messenger RNA expression in human bone marrow stromal cells

Blood ◽

10.1182/blood.v95.10.3094 ◽

2000 ◽

Vol 95 (10) ◽

pp. 3094-3101 ◽

Cited By ~ 63

Author(s):

Ranita Sungaran ◽

Orin T. Chisholm ◽

Boban Markovic ◽

Levon M. Khachigian ◽

Yoshihiro Tanaka ◽

...

Keyword(s):

Bone Marrow ◽

Growth Factor ◽

Mrna Expression ◽

Stromal Cells ◽

Messenger Rna ◽

Human Bone ◽

Human Bone Marrow ◽

Dependent Manner ◽

Platelet Factor ◽

Dose Dependent

Abstract Thrombopoietin (TPO), the specific cytokine that regulates platelet production, is expressed in human bone marrow (BM), kidney, and liver. There appears to be no regulation of TPO in the kidney and liver, but TPO messenger RNA (mRNA) expression can be modulated in the stromal cells of the BM. In this study, we used primary human BM stromal cells as a model to study the regulation of TPO mRNA expression in response to various platelet -granular proteins. We showed that platelet-derived growth factor (PDGF) BB and fibroblast growth factor (FGF) 2 stimulated TPO mRNA expression in both a dose-dependent and time-dependent manner. The addition of 50 ng/mL of PDGF and 20 ng/mL of FGF resulted in maximal induction of TPO mRNA expression in 4 hours. We also found that platelet factor 4 (PF4), thrombospondin (TSP), and transforming growth factor-beta (TGF-β) are negative modulators of megakaryocytopoiesis. We observed suppression in TPO mRNA expression with 1 μg/mL of both PF4 and TSP and 50 ng/mL of TGF-β, with maximal suppression occurring 4 hours after the addition of these proteins. Finally, the addition of whole-platelet lysate produced a dose-dependent inhibition of TPO expression. On the basis of these findings, we propose that the platelet -granular proteins studied may regulate TPO gene expression in BM stromal cells by means of a feedback mechanism.

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Bone Marrow-Derived MSCs Stimulated by IFN-γ Inhibited the Growth of ToxoplasmaGondii Via up-Regulation of GBP1

Blood ◽

10.1182/blood.v124.21.5143.5143 ◽

2014 ◽

Vol 124 (21) ◽

pp. 5143-5143

Author(s):

Aiping Qin ◽

De-Hua Lai ◽

Weijun Huang ◽

Mingshui Wu ◽

Xiaoyong Chen ◽

...

Keyword(s):

Bone Marrow ◽

Natural Science ◽

Research Funding ◽

Guangdong Province ◽

Human Mscs ◽

Dependent Manner ◽

Rna Seq ◽

Guangzhou City ◽

Ifn Γ ◽

Dose Dependent

Abstract Background Mesenchymal stromal cells (MSCs) are a heterogeneous cell population endowed with multi-lineage differentiation potential and extensive immunomodulatory properties. MSCs have been successfully used for prevention and treatment of immune disorders such as graft-versus-host disease. Emerging preclinical studies suggest that MSCs might also protect against infectious challenge. Aims This study aimed to rule out the potential mechanism of human MSCs against Toxoplasma gondii (T. gondii). Methods Human bone marrow-derived MSCs (hMSCs) were pretreated for 24h with a series of concentrations of IFN-γ and then infected with T. gondii strains of variant virulences (virulent RH and avirulent ME49). RNA-seq and westernblots were used to analyze gene and protein expression patterns of hMSCs in IFN-γ-stimulated and unstimulated conditions. The intracellular parasites (with fluorescence labeled) were counted microscopically at multiple time points postinfection. The short hairpin RNA (shRNA) expression was used to generate RNAi of GBP-1, GBP-2 and GBP-5. Results Human MSCs stimulated with IFN-γ were capable to inhibit the growth of T. gondii (eg: at IFN-γ 10ng/ml, the inhibition rates are 26.5% (RH) and 37.5% (ME49) 12hr postinfection) in a dose-dependent manner. Compared with the unstimulated MSCs (controls), IFN-γ treatment at 5, 10, 20ng/ml inhibited T. gondii (ME49) growth by percent of 27.1±7.9, 37.5±6.2, 47.0±7.6 (mean±SD, n=4) 12 hr postinfection and the inhibition rates are 54.5±2.1%, 62.5±4.9% and 78.5±2.1 at 24 hr postinfection, respectively. After 48 hr postinfection, the ratio between parasites per parasitophorous vacuole (PV) containing rosettes and single paraites in IFN-γ-stimulated MSCs was significantly reduced compared with that in the unstimulated MSCs (p<0.01, p<0.01, p<0.001 for ME49 at IFN-γ 5, 10, 20ng/ml, respectively). Furthermore, There was no significant effect of conditioned medium (CM) from IFN-γ-stimulated MSCs on T. gondii growth in comparison with CM from unstimulated MSCs (p=0.74 for RH and p=0.69 for ME49). We observed that the resistance in hMSCs does not depend on IDO (p=0.85 for RH and p=0.79 for ME49). RNA-seq data showed that IFN-γ-inducible p65 guanylate-binding proteins (GBPs) might play pivotal roles in the inhibition of T. gondii growth. Reads per kilobase-pairs per million (RPKM) mean values of GBP1, 2, 5 in IFN-γ-stimulated MSCs are 1093.3, 443.3, 348.2, respectively. By RNAi knockdown, the results showed that silencing of GBP1 (but not GBP2, GBP5) in hMSCs resulted in recovery of T. gondii growth inhibition at 12 hr and 24 hr postinfection (p<0.05 and p<0.001 for ME49). Conclusion: Human MSCs pre-stimulated with IFN-γ inhibited the growth of T. gondii in a dose-dependent manner via up-regulation of GBP-1 expression. Disclosures Liu: the project of the Zhujiang Science & Technology Star of Guangzhou city (2013027): Research Funding; the Technology Plan of Guangdong Province of China (2012B031800403): Research Funding; the project of health collaborative innovation of Guangzhou city (201400000003-4, 201400000003-1): Research Funding; Natural Science Foundation of Guangdong Province (S2012010009299): Research Funding; National Public Health Grand Research Foundation (201202017): Research Funding; National High Technology Research and Development Program of China (863 Program) (2011AA020105): Research Funding; National Natural Science Foundation of China (81270647, 81300445, 81200388): Research Funding.

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