Stimulation of immune suppressive CD34 + cells from normal bone marrow by Lewis lung carcinoma tumors

1998 ◽  
Vol 46 (5) ◽  
pp. 253-260 ◽  
Author(s):  
Mark A. Wright ◽  
Kristina Wiers ◽  
Kishore Vellody ◽  
Dragana Djordjevic ◽  
M. Rita I. Young
Keyword(s):  
Bone Marrow ◽  
Lung Carcinoma ◽  
Lewis Lung Carcinoma ◽  
Normal Bone Marrow ◽  
Lewis Lung ◽  
Normal Bone ◽  
Cd34 Cells ◽  
Stimulation Of

scholarly journals Cytokines increase human hemopoietic cell adhesiveness by activation of very late antigen (VLA)-4 and VLA-5 integrins.

1995 ◽  
Vol 181 (5) ◽  
pp. 1805-1815 ◽  
Author(s):  
J P Lévesque ◽  
D I Leavesley ◽  
S Niutta ◽  
M Vadas ◽  
P J Simmons
Keyword(s):  
Bone Marrow ◽  
Progenitor Cells ◽  
Normal Bone Marrow ◽  
Gm Csf ◽  
Normal Bone ◽  
Cd34 Cells ◽  
Late Antigen ◽  
Beta 1 Integrin ◽  
Functional States ◽  
Adhesive Interactions

Cytokines are known to be important regulators of normal hemopoiesis, acting in concert with components of the bone marrow microenvironment. Interactions with this microenvironment are known to regulate the proliferation, differentiation, and homing of hemopoietic progenitor (CD34+) cells. Adhesive interactions with the extracellular matrix retain CD34+ cells in close proximity to cytokines, but may also provide important costimulatory signals. Thus, the functional states of adhesion receptors are critical properties of CD34+ cells, but the physiological mechanisms responsible for regulating functional properties of cell adhesion receptors on primitive hemopoietic cells are still unknown. We confirm that the integrins very late antigen (VLA)-4 and VLA-5 are expressed on the CD34+ cell lines MO7e, TF1, and on normal bone marrow CD34+ progenitor cells, but in a low affinity state, conferring on them a weak adhesive phenotype on fibronectin (Fn). Herein, we show that the cytokines interleukin (IL)-3, granulocyte-macrophage CSF (GM-CSF), and KIT ligand (KL) are physiological activators of VLA-4 and VLA-5 expressed by MO7e, TF1, and normal bone marrow CD34+ progenitor cells. Cytokine-stimulated adhesion on Fn is dose dependent and transient, reaching a maximum between 15 and 30 min and returning to basal levels after 2 h. This cytokine-dependent activation is specific for VLA-4 and VLA-5, since activation of other beta 1 integrins was not observed. The addition of second messenger antagonists staurosporine and W7 abolished all cytokine-stimulated adhesion to Fn. In contrast, genistein inhibited KL-stimulated adhesion, but failed to inhibit GM-CSF- and IL-3-stimulated adhesion. Our data suggest that cytokines GM-CSF and IL-3 specifically stimulate beta 1 integrin function via an "inside-out" mechanism involving protein kinase activity, while KL stimulates integrin activity through a similar, but initially distinct, pathway via the KIT tyrosine-kinase. Thus, in addition to promoting the survival, proliferation, and development of hemopoietic progenitors, cytokines also regulate adhesive interactions between progenitor cells and the bone marrow microenvironment by modifying the functional states of specific integrins. These data are of importance in understanding the fundamental processes of beta 1 integrin activation and cellular response to mitogenic cytokines as well as on the clinical setting where cytokines induce therapeutic mobilization of hematopoietic progenitors.

Role for PP2A Regulatory Subunit B55α as A Regulator of AKT and Other Signaling Pathways In AML.

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 1668-1668
Author(s):  
Peter P. Ruvolo ◽  
YiHua Qui ◽  
Kevin R Coombes ◽  
Nianxiang Zhang ◽  
Vivian Ruvolo ◽  
...  
Keyword(s):  
Gene Expression ◽  
Bone Marrow ◽  
Protein Expression ◽  
Normal Bone Marrow ◽  
Protein Levels ◽  
B Subunit ◽  
Normal Bone ◽  
Cd34 Cells ◽  
Blast Cells ◽  
B Subunits

Abstract Abstract 1668 Activation of survival kinases such as Protein Kinase B (AKT), Protein Kinase C (PKC), and Extracellular Receptor Activated Kinase (ERK) predict poor clinical outcome for patients with acute myeloid leukemia (AML; Kornblau et al Blood 2006). A better understanding of how the activities of these kinases are regulated by phosphorylation and dephosphorylation will enable the development of targeted therapies directed against this axis. Protein Phosphatase 2A (PP2A) negatively regulates PKC, AKT, and ERK but its role in AML is not clear. In the current study we examined the role of PP2A in regulating AKT in AML. Activation of AKT involves phosphorylation of threonine 308 (T308) and serine 473 (S473). A recent study has indicated that phosphorylation of AKT at T308 but not S473 is a poor prognostic factor for AML patients and that PP2A activity negatively correlated with T308 phosphorylation (Gallay et al Leukemia 2009). PP2A is a family of different isoforms that form hetero-trimers consisting of a catalytic C subunit, a scaffold A subunit, and one of at least 21 different regulatory B subunits. The functionality of each PP2A isoform is determined by the regulatory B subunit. Thus to understand PP2A regulation of AKT in AML, it is essential to study the B subunit that regulates the AKT phosphatase. The PP2A isoform regulating AKT in the AML patients is currently unknown. Evidence suggests that the B55a subunit is responsible for dephosphorylation of AKT at T308. In the current study, we compared B55α gene expression in blast cells derived from AML patients with normal counterpart (i.e. CD34+) cells derived from normal bone marrow donors by real time PCR. Surprisingly, B55α gene expression was higher in the patients. Reverse Phase Protein Analysis (RPPA) is a powerful tool that allows for the analysis of protein expression from patient samples. Protein levels of the PP2A B subunit were analyzed by RPPA in AML blast cells obtained from 511 newly diagnosed AML patients and CD34+ cells obtained from 11 normal bone marrow donors. Levels of B55α protein were significantly lower in the blast cells from the AML patients compared to normal CD34+ cells. While the mechanism for the observed difference in gene versus protein expression in the leukemia cells has yet to be determined, a plausible mechanism is that the B55α protein is being proteolyzed since monomeric PP2A B subunits that are not part of the PP2A hetero-trimer are degraded. Importantly the reduced levels of B55α protein observed would be predicted if AKT were activated in the AML blast cells. We next compared AKT phosphorylation status with B55α protein expression in the AML blast cells using RPPA to answer this question. Analysis of RPPA data revealed that there was no correlation between B55α protein levels and levels of total AKT protein or with levels of AKT phosphorylated at S473 in the AML samples. However, there was a moderate but significant negative correlation between B55α protein levels and levels of AKT phosphorylated at T308. This result suggests that B55α is mediating dephosphorylation of AKT at T308 but not S473 in the AML cells. B55α expression was not associated with FAB classification but was positively correlated with high blast and peripheral blood counts. While the level of expression of the B subunit did not correlate with overall survival, intermediate levels of B55α expression were associated with longer complete remission duration. We predict that higher levels of B55α would reflect low levels of other PP2A B subunits. Consistent with this prediction, B55α expression positively correlated with MYC expression in the AML patients. MYC expression is regulated by a B subunit that competes with B55α (i.e. B56α). These findings suggest that B55α may play an important role in AML as a negative regulator of AKT and perhaps by other as yet unidentified functions. Activation of B55α is a potential therapeutic target for overcoming the AKT activation frequently observed in AML. Disclosures: No relevant conflicts of interest to declare.

Gene expression in CD34+ cells from normal bone marrow and leukemic origins

2000 ◽  
Vol 1 (3) ◽  
pp. 206-217 ◽  
Author(s):  
Jian Gu ◽  
Qing-Hua Zhang ◽  
Qiu-Hua Huang ◽  
Shuang-Xi Ren ◽  
Xin-Yan Wu ◽  
...  
Keyword(s):  
Gene Expression ◽  
Bone Marrow ◽  
Normal Bone Marrow ◽  
Normal Bone ◽  
Cd34 Cells

Apoptosis-Independent Activation of Caspase-3 in CD34–Positive (CD34+) Mobilized Cells.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 2305-2305
Author(s):  
Karine Augeul-Meunier ◽  
Carine Crampé ◽  
Philippe Farce ◽  
Christiane Mounier ◽  
Denis Guyotat ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Peripheral Blood ◽  
Caspase 3 ◽  
Normal Bone Marrow ◽  
Hematopoietic Stem ◽  
Normal Bone ◽  
Peripheral Blood Cd34 ◽  
Cd34 Cells ◽  
Significant Difference ◽  
Mobilized Peripheral Blood

Abstract G-CSF mobilized peripheral blood CD34+ cells are now the preferred and major source of hematopoietic stem and progenitor cells harvested for both autologous and allogeneic transplantation. Several mechanisms, like SDF-1/CXCR4 interactions or degradation of adhesion molecules by proteolytic environnement, are involved in the mobilization process. However this phenomenon is still partially understood. Gene expression analysis has identified an overexpression of the caspase-3 gene in CD34+ mobilized cells, compared to CD34+ from normal bone marrow. Caspase-3 is the main effector of the terminal phase of apoptosis. However recent studies have provided evidence of its implication in non apoptotic cellular processes, such as differentiation, migration and cytoskeleton modelling. We evaluated by multicolour flow cytometry the expression of activated caspase-3 in G-CSF mobilized CD34+/CD45+ cells from blood (n=16), and from apheresis products (n=10). CD34+/CD45+ cells from normal bone marrow (n=4) served as control. Caspase-3 activity on fluorescent substrate (PhiPhiLux method) and apoptosis (Annexin V assay) were also evaluated. Finally we analysed the expression of anti apoptotic proteins Bcl-2, Bcl-Xl, and of Heat Shock Proteins HSP27, HSP70 and HSP90 in the same cell population. There was no significant difference for apoptosis between mobilized and bone marrow CD34+ cells (26% versus 33% apoptotic cells). Activated caspase-3 levels were significantly higher in mobilized CD34+ cells (mean fluorescence intensity 3.64 fold higher). This was consistent with cleavage of caspase-3 substrate observed in mobilized cells, but not in bone marrow CD34+ cells. An increased expression of HSP90 (of which caspase-3 is a client protein) was observed in peripheral CD34+ cells, but there was no variation of BCl-2 and Bcl-Xl expression. Our results show an activation of caspase-3 in the mobilized peripheral blood CD34+ cells, which appears to be independent of apoptosis induction. The role of this activation and possible control by HSPs warrants further analysis to establish its relationship with mobilization mechanisms.

The Proliferation Inhibitor CDKN1C (P57KIP2) Is Over Expressed in CD34+ Cells of Patients with MDS and Determines a Worse Prognosis Independently of IPSS Score Factors

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 3820-3820
Author(s):  
Sascha Dietrich ◽  
Mindaugas Andrulis ◽  
Andrea Pellagatti ◽  
Aleksandar Radujkovic ◽  
Ulrich Germing ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Overall Survival ◽  
Protein Expression ◽  
Normal Bone Marrow ◽  
Double Staining ◽  
Expression Levels ◽  
Cyclin Dependent Kinases ◽  
Normal Bone ◽  
Cd34 Cells ◽  
Worse Prognosis

Abstract Abstract 3820 Purpose: Progressive cytopenias are the cause of death for the majority of patients with myelodysplastic syndromes (MDS). In order to investigate if the proliferative activity of CD34+ cells in MDS bone marrow correlates with prognosis, we measured expression levels of the proliferation inhibitor CDKN1C (P57KIP2) in two independent study cohorts. Patients and methods: Gene expression profiling data on bone marrow CD34+ cells were obtained from 183 MDS patients (55 with RA, 48 with RARS, 37 with RAEB1 and 43 with RAEB2). mRNA expression levels of CDKN1C (P57KIP2) were correlated with overall survival (OS) and proliferation markers such as PCNA, cyclins and cyclin dependent kinases. In a second independent patient cohort comprising 93 patients (17 had RA, 13 REAB1, 27 RAEB2, 29 AML arising from MDS and 7 CMML), protein expression levels of CDKN1C (P57KIP2) were evaluated by immune histochemistry (IHC) in trephine biopsies. Protein expression levels of CDKN1C (P57KIP2) were correlated with clinical outcome, OS and the proliferation marker KI67 in CD34+ cells (double staining). Results: In purified CD34+ cells, mRNA expression of CDKN1C (P57KIP2) correlated with higher risk and poorer overall survival of patients with MDS (p=0.0006, n=183, Figure1). Furthermore, increased CDKN1C (P57KIP2) expression was significantly associated with loss of CD38 (p=0.002) as well as loss of proliferating cell nuclear antigen (PCNA, p<0.001), cyclins and cyclin-dependent kinases (p<0.001) underlining the role of CDKN1C (P57KIP2) as a proliferation inhibitor. Similarly, protein expression of CDKN1C (P57KIP2) determined in trephine biopsies predicted a poor prognosis of patients with MDS (p=0.0003, HR=2.2, n=93, Figure 1). No expression of CDKN1C (P57KIP2) could be demonstrated in 10 control patients with normal bone marrow. Separate evaluation of WHO risk categories revealed that in patients with less than 10% blasts (RA, RAEB1 and CMML1), CDKN1C (P57KIP2) was not predictive for OS. In contrast, patients with high CDKN1C (P57KIP2) expression and RAEB2 (p=.0.02, HR 2.4) or AML arising from MDS (p=0.002, HR 2.9) had a significantly worse OS than patients with low CDKN1C (P57KIP2) levels. CDKN1C (P57KIP2) expression analysis within the group of allogeneic stem cell recipients showed no impact on survival after transplant. Multivariate cox regression analysis with the confounding co-variates: age, IPSS score factors (cytopenias, cytogenetic risk profile, blast count) and primary versus secondary MDS confirmed the independent impact of CDKN1C (P57KIP2) expression on OS (p=0.0002, HR 2.9). CDKN1C (P57KIP2) protein expression could also be demonstrated in sorted CD34+ cells of MDS patients by western blot analysis and was significantly higher than in CD34- cells (p=0.03, n=7). KI67 expression was evaluated in CD34+ cells by IHC double staining in 34 trephine biopsies and 10 control patients with normal bone marrow. Percentages of CD34+ and KI67 positive cells were higher in control patients and patients with low risk MDS (RA, RAEB1) than in patients with high risk MDS (RAEB2) (p<0.01). Patients with AML (>20% blasts) had significantly higher levels of KI67 (p<0.05). In patients with MDS (<20% blasts) KI67 percentages correlated inversely with CDKN1C (P57KIP2) expression (p=0.04). Conclusions: High CDKN1C (P57KIP2) expression in CD34+ cells of patients with MDS is associated with a reduced fraction of proliferative CD34+ cells and determines a worse prognosis independently of factors used to calculate the IPSS score. Allogeneic stem cell transplantation could overcome the worse prognosis of high CDKN1C (P57KIP2) expression. Further studies are warranted to determine the impact of CDKN1C (P57KIP2) expression to guide clinical decisions. Disclosures: No relevant conflicts of interest to declare.

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