KCa3.1 Blockers Inhibit Cell Proliferation in Chronic Lymphocytic Leukemia

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 3887-3887
Author(s):  
Eva M Groessinger ◽  
Lukas Weiss ◽  
Elisabeth Hinterseer ◽  
Judith Schmoelzer ◽  
Karin Oberascher ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
T Cells ◽  
Chronic Lymphocytic Leukemia ◽  
Peripheral Blood ◽  
Lymphocytic Leukemia ◽  
K Channel ◽  
Relative Reduction ◽  
Rt Pcr ◽  
Ki 67

Abstract Abstract 3887 Potassium (K)-channels play an important role in regulating cell proliferation by maintenance of the membrane potential and subsequent Ca2+ signaling. Out of 80 known human K-channel genes only the voltage gated K-channel Kv1.3 and the calcium-gated K-channel KCa3.1 are expressed in lymphocytes, with expression levels varying greatly depending on lymphocyte maturation and activation status (for review see Cahalan MD, Chandy KG, Immunol Rev. 2009). Accordingly, proliferation of various lymphocyte subtypes can be inhibited by blockade of the respective predominant K-channel. As the modulation of K-channel expression on malignantly transformed lymphocytes and their potential as therapeutic targets has been largely overlooked, we characterized the expression and function of Kv1.3 and KCa3.1 in Chronic Lymphocytic Leukemia (CLL). Primary cells from unselected CLL-patients were isolated from peripheral blood mononuclear cells (PBMCs). Comparison of Kv1.3 and KCa3.1 levels on unstimulated CLL-cells versus PMA/ionomycin-activated CLL-cells revealed a significant reduction in the Kv1.3/KCa3.1 ratio (n=31, 1.526 vs. 0.9054, p=0.0005), as evidenced on mRNA and protein levels by RT-PCR and patch clamp analysis, respectively. Stimulation of CLL-cells with enriched and activated autologous CD4+ T-cells resulted in higher CLL-cell activation as measured by CD80/86 expression, and an even more pronounced reduction of the Kv1.3/KCa3.1 ratio. This stimulation protocol also effectively induced CLL-cell proliferation as verified by Ki-67 expression and CFSE dilution via flow cytometric measurement. Highly activated and/or proliferating CLL-cells consistently up-regulated KCa3.1 (RT-PCR: Ki-67 n=5, p=0.0013; CFSE n=5, p=0.0436; immunofluorescence (IF) staining - n=4, p=0.0121), whereas Kv1.3 was fairly low. Consistent with our in vitro data, CLL-cells in lymph node and bone marrow, believed to be primary sites of CLL-proliferation in vivo, were highly positive for KCa3.1 channels in contrast to CLL-cells from the peripheral blood as revealed by IF staining on paraffin-embedded tissue sections. In light of the significantly increased KCa3.1-expression on activated and proliferating CLL-cells, we investigated whether specific blockade of KCa3.1 could inhibit CLL-cell proliferation. CLL-cells in PBMCs were pre-stimulated by co-culture with CD40L expressing (murine) fibroblasts for 24 hours, and were treated with highly specific blockers for KCa3.1 (TRAM-34 or Clotrimazole) or Kv1.3 (PAP-1 or Psora-4) prior to addition of α-CD3/CD28 beads to activate autologous T-cells. KCa3.1 blockade could effectively diminish CLL-cell cycle entry in all samples investigated (TRAM-34: n=12, p<0.0001; Clotrimazole: n=5, p=0.0625) with a mean relative reduction in Ki-67+ cells of 52% (SD=1.56) for TRAM-34 (see Figure left) and 53% (SD=1.11) for Clotrimazole. This antagonizing effect of TRAM-34 was clearly dose-dependent (n=10) without affecting CLL-cell viability (see Figure right) nor activation. Viability, activation and proliferation of T-cells and fibroblasts present in the co-culture system were not affected by TRAM-34. Blockade of Kv1.3 did not reduce proliferation in CLL-cells. In summary, we showed that CLL-cells exhibit significant changes in their K-channel constitution following activation and proliferation, analogous to healthy B-cells. Notably, in vitro CLL-cell proliferation was effectively inhibited by highly specific KCa3.1 blockers. Thus far in vitro testing of potential CLL-drugs has been primarily performed on G0-arrested CLL-cells, sometimes in co-culture with stromal cells to enhance their viability. Our approach attempts to specifically target proliferating CLL-cells, presumably the most relevant CLL-cell fraction contributing to disease progression. Given their low toxicity profile, KCa3.1 blockers could represent a promising therapeutic option in CLL. Disclosures: No relevant conflicts of interest to declare.

Increased CD39 Expression on CD4+ T-Lymphocytes Has Clinical and Prognostic Significance in Chronic Lymphocytic Leukemia,

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 3895-3895
Author(s):  
Yair Herishanu ◽  
Inbal Hazan-Hallevi ◽  
Sigi Kay ◽  
Varda Deutsch ◽  
Aaron Polliack ◽  
...  
Keyword(s):  
T Cells ◽  
Chronic Lymphocytic Leukemia ◽  
T Lymphocytes ◽  
Peripheral Blood ◽  
Conflicts Of Interest ◽  
Prognostic Significance ◽  
Lymphocytic Leukemia ◽  
Leukemic Cells ◽  
Anti Inflammatory

Abstract Abstract 3895 Chronic lymphocytic leukemia (CLL) cells depend on their microenvironment for proliferation and survival. Ectonucleotidase CD39 has anti-inflammatory properties as it hydrolyzes pro-inflammatory extra-cellular ATP, generates anti-inflammatory adenosine and also protects regulatory T cells from ATP-induced cell death. In this study we investigated the clinical significance of CD39 expression on CD4+T-cells in 45 patients with CLL as well as its compartmental regulation and explored the possible mechanisms for its induction. Compared to healthy individuals, CD4+CD39+ lymphocytes were increased in the peripheral blood of patients with CLL (4.6%±2.28 vs. 17.3%±12.49, respectively, p=0.004), and correlated with advanced stage of disease (9.72%±5.76, 18.15%±12.03 and 25.90%±16.34, of CD4+ lymphocytes, in patients with Rai stages 0, 1+2 and 3+4, respectively, p=0.019). CD4+CD39+ cells were also higher in patients with CLL who needed therapeutic intervention (untreated; 12.99%±10.63 vs treated; 22.21%±12.88, p=0.01) and in those who were ZAP70+ or had b2-microglobulin levels>3g/L. There were more CD4+CD39+ lymphocytes in the bone marrow compartment (22.25%±16.16) than in the peripheral blood (16.60%±15.84, p=0.009). In-vitro studies showed that CD39 can be induced on CD4+cells by exposure to ATP or indirectly, following B-cell receptor (BCR) engagement (CD4+CD39+ lymphocytes increased by 1.56 fold, in the BCR engaged samples compared to their paired controls; 20.27%±11.3 vs. 13%±9.42, respectively, p=0.0006). Conclusions: Increased CD39 expression on CD4+ T-lymphocytes in CLL associates with an aggressive disease. This may reflect the ability of the leukemic cells to suppress the surrounding immune environment, and contribute to a poorer prognosis. CD39+ may also serve as a future target for the development of novel therapies with immune modulating anti–tumor agents in CLL. Disclosures: No relevant conflicts of interest to declare.

Differential Effects of Microenvironmental Elements On Tumor Cells Survival in Chronic Lymphocytic Leukemia Patient Subsets with Good or Poor Prognosis.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 2333-2333
Author(s):  
Marta Coscia ◽  
Francesca Pantaleoni ◽  
Chiara Riganti ◽  
Micol Rigoni ◽  
Candida Vitale ◽  
...  
Keyword(s):  
T Cells ◽  
Chronic Lymphocytic Leukemia ◽  
Tumor Cells ◽  
Peripheral Blood ◽  
Lymphocytic Leukemia ◽  
Leukemic Cells ◽  
Spontaneous Apoptosis ◽  
Survival Effect

Abstract Abstract 2333 Poster Board II-310 Chronic lymphocytic leukemia (CLL) is a clinically heterogeneous disease. A very reliable prognosticator is the mutational status of the tumor immunoglobulin heavy chain variable region (IgVH): patients with unmutated (UM) IgVH have a worse prognosis than patients with mutated (M) IgVH. Soluble factors (i.e. IL-4 and CD40L) and cellular components of the local microenvironment [i.e. bone marrow stromal cells (BMSC) and nurse-like cells (NLCs)] are important survival factors for CLL B cells. It is currently unknown to what extent UM and M CLL cells depend on the local microenvironment for their survival. We have evaluated the spontaneous apoptotic rate of tumor cells isolated by immunomagnetic selection from the peripheral blood (PB) of M and UM CLL patients. Leukemic cells purified by negative selection from the PB of UM CLL patients showed significantly higher rates of spontaneous apoptosis after long-term in vitro culture as compared to CLL cells isolated from M patients. Both M and UM CLL cells showed high basal level of Bcl-2 expression and NF-kB activity soon after purification. In vitro spontaneous apoptosis of purified UM CLL cells was associated with a progressive downregulation of the intracellular expression of Bcl-2 and with a complete loss of the active nuclear form of NF-kB. On the contrary, the higher long term viability of M CLL cells was paralleled by a maintained Bcl-2 and NF-kB expression. IL-4 and CD40L, used alone or in combination, as well as murine and human BMSC were capable of rescuing UM tumor cells from apoptosis. The pro-survival effect of these stimuli was exerted through the upregulation of Bcl-2 and was totally independent from the recovery of NF-kB nuclear translocation. We observed that UM CLL cells were less susceptible to spontaneous apoptosis when cultured as unfractionated peripheral blood mononuclear cells (PBMC) as compared to purified leukemic cells. This higher cell viability was associated with a retained expression of Bcl-2 and of the nuclear form of NF-kB, thus suggesting the presence of a pro-survival element in the peripheral blood of these patients. The extremely low numbers of NLCs generated from PMBC of UM patients ruled out a role for these cells in supporting the survival of unpurified leukemic cells. Conversely, a pro-survival effect on UM CLL cells was exerted by autologous T cells. Indeed, a significant reduction in the apoptotic rate of leukemic cells was observed when purified UM cells were cultured in the presence of autologous peripheral blood T cells (PBT). The prosurvival effect of circulating T cells was particularly evident at high T:B ratio, did not require a cell-cell contact and was mediated by the upregulation of Bcl-2 and the activation of NF-kB in leukemic cells. These data indicate that the survival of UM tumor cells is highly dependent on the action of multiple microenviromental stimuli. Conversely, M cells are intrinsically more resistant to apoptosis and minimally influenced by the local microenviroment. The higher dependency of UM CLL cells from extrinsic signals might be exploited to develop new therapies targeting the tumor microenvironment and to improve the outcome of more aggressive CLL. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Immunomodulatory effects of different intravenous immunoglobulin preparations in chronic lymphocytic leukemia

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Ana Colado ◽  
Esteban Enrique Elías ◽  
Valeria Judith Sarapura Martínez ◽  
Gregorio Cordini ◽  
Pablo Morande ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
B Cells ◽  
Chronic Lymphocytic Leukemia ◽  
T Cell Activation ◽  
Cell Activation ◽  
Lymphocytic Leukemia ◽  
Immunomodulatory Effects ◽  
Immunoglobulin Preparations

AbstractHypogammaglobulinemia is the most frequently observed immune defect in chronic lymphocytic leukemia (CLL). Although CLL patients usually have low serum levels of all isotypes (IgG, IgM and IgA), standard immunoglobulin (Ig) preparations for replacement therapy administrated to these patients contain more than 95% of IgG. Pentaglobin is an Ig preparation of intravenous application (IVIg) enriched with IgM and IgA (IVIgGMA), with the potential benefit to restore the Ig levels of all isotypes. Because IVIg preparations at high doses have well-documented anti-inflammatory and immunomodulatory effects, we aimed to evaluate the capacity of Pentaglobin and a standard IVIg preparation to affect leukemic and T cells from CLL patients. In contrast to standard IVIg, we found that IVIgGMA did not modify T cell activation and had a lower inhibitory effect on T cell proliferation. Regarding the activation of leukemic B cells through BCR, it was similarly reduced by both IVIgGMA and IVIgG. None of these IVIg preparations modified spontaneous apoptosis of T or leukemic B cells. However, the addition of IVIgGMA on in vitro cultures decreased the apoptosis of T cells induced by the BCL-2 inhibitor, venetoclax. Importantly, IVIgGMA did not impair venetoclax-induced apoptosis of leukemic B cells. Overall, our results add new data on the effects of different preparations of IVIg in CLL, and show that the IgM/IgA enriched preparation not only affects relevant mechanisms involved in CLL pathogenesis but also has a particular profile of immunomodulatory effects on T cells that deserves further investigation.

scholarly journals CD73+ CD127high Long-Term Memory CD4 T Cells Are Highly Proliferative in Response to Recall Antigens and Are Early Targets in HIV-1 Infection

2021 ◽  
Vol 22 (2) ◽  
pp. 912
Author(s):  
Nabila Seddiki ◽  
John Zaunders ◽  
Chan Phetsouphanh ◽  
Vedran Brezar ◽  
Yin Xu ◽  
...  
Keyword(s):  
T Cells ◽  
Peripheral Blood ◽  
Cd4 T Cells ◽  
Significant Proportion ◽  
Long Term Memory ◽  
Ki 67 ◽  
Memory Cd4 T Cells ◽  
Hiv 1

HIV-1 infection rapidly leads to a loss of the proliferative response of memory CD4+ T lymphocytes, when cultured with recall antigens. We report here that CD73 expression defines a subset of resting memory CD4+ T cells in peripheral blood, which highly express the α-chain of the IL-7 receptor (CD127), but not CD38 or Ki-67, yet are highly proliferative in response to mitogen and recall antigens, and to IL-7, in vitro. These cells also preferentially express CCR5 and produce IL-2. We reasoned that CD73+ memory CD4+ T cells decrease very early in HIV-1 infection. Indeed, CD73+ memory CD4+ T cells comprised a median of 7.5% (interquartile range: 4.5–10.4%) of CD4+ T cells in peripheral blood from healthy adults, but were decreased in primary HIV-1 infection to a median of 3.7% (IQR: 2.6–6.4%; p = 0.002); and in chronic HIV-1 infection to 1.9% (IQR: 1.1–3%; p < 0.0001), and were not restored by antiretroviral therapy. Moreover, we found that a significant proportion of CD73+ memory CD4+ T cells were skewed to a gut-homing phenotype, expressing integrins α4 and β7, CXCR3, CCR6, CD161 and CD26. Accordingly, 20% of CD4+ T cells present in gut biopsies were CD73+. In HIV+ subjects, purified CD73+ resting memory CD4+ T cells in PBMC were infected with HIV-1 DNA, determined by real-time PCR, to the same level as for purified CD73-negative CD4+ T cells, both in untreated and treated subjects. Therefore, the proliferative CD73+ subset of memory CD4+ T cells is disproportionately reduced in HIV-1 infection, but, unexpectedly, their IL-7 dependent long-term resting phenotype suggests that residual infected cells in this subset may contribute significantly to the very long-lived HIV proviral DNA reservoir in treated subjects.

Evaluation of vecabrutinib as a model for non-covalent BTK/ITK inhibition for treatment of chronic lymphocytic leukemia

Blood ◽  
2021 ◽  
Author(s):  
Billy Michael Chelliah Jebaraj ◽  
Annika Müller ◽  
Rashmi Priyadharshini Dheenadayalan ◽  
Sascha Endres ◽  
Philipp M. Roessner ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Chronic Lymphocytic Leukemia ◽  
Inhibitory Effect ◽  
Lymphocytic Leukemia ◽  
Transfer Model ◽  
Treatment Benefit ◽  
Btk Inhibitors

Covalent Bruton tyrosine kinase (BTK) inhibitors such as ibrutinib have proven to be highly beneficial in the treatment of chronic lymphocytic leukemia (CLL). Interestingly, the off-target inhibition of IL-2-inducible T-cell kinase (ITK) by ibrutinib may also play a role in modulating the tumor microenvironment, potentially enhancing the treatment benefit. However, resistance to covalently binding BTK inhibitors can develop by a mutation in cysteine 481 of BTK (C481S), which prevents the irreversible binding of the drugs. In the present study we performed pre-clinical characterization of vecabrutinib, a next generation non-covalent BTK inhibitor, with ITK inhibitory properties similar to those of ibrutinib. Unlike ibrutinib and other covalent BTK inhibitors, vecabrutinib showed retention of the inhibitory effect on C481S BTK mutants in vitro, similar to that of wildtype BTK. In the murine Eµ-TCL1 adoptive transfer model, vecabrutinib reduced tumor burden and significantly improved survival. Vecabrutinib treatment led to a decrease in CD8+ effector and memory T-cell populations, while the naïve populations were increased. Of importance, vecabrutinib treatment significantly reduced frequency of regulatory CD4+ T-cells (Tregs) in vivo. Unlike ibrutinib, vecabrutinib treatment showed minimal adverse impact on activation and proliferation of isolated T-cells. Lastly, combination treatment of vecabrutinib with venetoclax was found to augment treatment efficacy, significantly improve survival and lead to favourable reprogramming of the microenvironment in the murine Eµ-TCL1 model. Thus, non-covalent BTK/ITK inhibitors such as vecabrutinib may be efficacious in C481S BTK mutant CLL, while preserving the T-cell immunomodulatory function of ibrutinib.

scholarly journals Defective T cell-mediated, isotype-specific immunoglobulin regulation in B cell chronic lymphocytic leukemia

Blood ◽  
1988 ◽  
Vol 71 (4) ◽  
pp. 1012-1020 ◽  
Author(s):  
JS Moore ◽  
MB Prystowsky ◽  
RG Hoover ◽  
EC Besa ◽  
PC Nowell
Keyword(s):  
T Cells ◽  
T Cell ◽  
B Cells ◽  
Chronic Lymphocytic Leukemia ◽  
B Cell ◽  
Cell Activity ◽  
Lymphocytic Leukemia ◽  
Specific Immunoglobulin ◽  
Cell Chronic Lymphocytic Leukemia

The consistent occurrence of T cell abnormalities in patients with B cell chronic lymphocytic leukemia (B-CLL) suggest that the non- neoplastic host T cells may be involved in the pathogenesis of this B cell neoplasm. Because potential defects of immunoglobulin regulation are evident in B-CLL patients, we investigated one aspect of this by studying the T cell-mediated immunoglobulin isotype-specific immunoregulatory circuit in B-CLL. The existence of class-specific immunoglobulin regulatory mechanisms mediated by Fc receptor-bearing T cells (FcR + T) through soluble immunoglobulin binding factors (IgBFs) has been well established in many experimental systems. IgBFs can both suppress and enhance B cell activity in an isotype-specific manner. We investigated the apparently abnormal IgA regulation in a B-CLL patient (CLL249) whose B cells secrete primarily IgA in vitro. Enumeration of FcR + T cells showed a disproportionate increase in IgA FcR + T cells in the peripheral blood of this patient. Our studies showed that the neoplastic B cells were not intrinsically unresponsive to the suppressing component of IgABF produced from normal T cells, but rather the IgABF produced by the CLL249 host T cells was defective. CLL249 IgABF was unable to suppress IgA secretion by host or normal B cells and enhanced the in vitro proliferation of the host B cells. Size fractionation of both normal and CLL249 IgABF by gel-filtration high- performance liquid chromatography (HPLC) demonstrated differences in the ultraviolet-absorbing components of IgABF obtained from normal T cells v that from our patient with defective IgA regulation. Such T cell dysfunction may not be restricted to IgA regulation, since we have found similar expansion of isotype-specific FcR + T cells associated with expansion of the corresponding B cell clone in other patients with B-CLL. These data suggest that this T cell-mediated regulatory circuit could be significantly involved in the pathogenesis of B-CLL.

scholarly journals T cell chronic lymphocytic leukemia: presence in bone marrow and peripheral blood of cells that suppress erythropoiesis in vitro

Blood ◽  
1978 ◽  
Vol 52 (1) ◽  
pp. 255-260 ◽  
Author(s):  
R Hoffman ◽  
S Kopel ◽  
SD Hsu ◽  
N Dainiak ◽  
ED Zanjani
Keyword(s):  
Bone Marrow ◽  
T Cell ◽  
Chronic Lymphocytic Leukemia ◽  
T Lymphocytes ◽  
Peripheral Blood ◽  
Lymphocytic Leukemia ◽  
Transfusion Therapy ◽  
Cell Chronic Lymphocytic Leukemia ◽  
Marrow Cells

Abstract The pathogenesis of the anemia associated with malignancy was investigated in a patient with T cell chronic lymphocytic leukemia. The plasma clot culture system was used as a measure in vitro of erythropoiesis. The patient's peripheral blood and marrow T lymphocytes obtained both before and after transfusion therapy suppressed erythroid colony formation by normal human bone marrow cells. Pretreatment of the patient's bone marrow T cells by antithymocyte globulin (ATG) and complement reversed this suppression. In addition, pretreatment of the patient's marrow cells with ATG and complement markedly augmented erythropoiesis in vitro. The expression of erythroid activity caused by the selective destruction of the suppressor T lymphocytes in the patient's bone marrow with ATG and the suppression of normal erythropoiesis by the patient's bone marrow and peripheral blood lymphocytes suggest that interaction between the malignant T cell and the erythropoietin-responsive stem cell is important in production of anemia in this patient.

scholarly journals Defective interleukin-2 production and responsiveness by T cells in patients with chronic lymphocytic leukemia of B cell variety

Blood ◽  
1986 ◽  
Vol 67 (2) ◽  
pp. 279-284 ◽  
Author(s):  
O Ayanlar-Batuman ◽  
E Ebert ◽  
SP Hauptman
Keyword(s):  
Cell Proliferation ◽  
T Cells ◽  
T Cell ◽  
Chronic Lymphocytic Leukemia ◽  
B Cell ◽  
Proliferative Response ◽  
Interleukin 2 ◽  
Lymphocytic Leukemia ◽  
T Cell Proliferation ◽  
Activated T Cells

Abstract The present studies were designed to investigate the mechanism(s) of the defective T cell proliferative response to various stimuli in patients with B cell chronic lymphocytic leukemia B-CLL. In 14 patients with advanced B-CLL (stage III or IV) we found the T cell response in the autologous (auto) and allogeneic (allo) mixed lymphocyte reaction (MLR) to be 35.7% and 30% of the controls, respectively. Proliferation in the MLR depends upon the production of and response to interleukin 2 (IL 2), a T cell growth factor. IL 2 production in eight B-CLL patients was 22% of the control. The response to IL 2 was measured by the increase in the T cell proliferation in the MLR with the addition of IL 2. T cell proliferation in both the auto and allo MLR of CLL patients was significantly lower than in the controls after the addition of IL 2. The proliferative response of normal T cells to stimulation by CLL B cells was 50% of the control. This latter response was increased to control levels when cultures were supplemented with exogenous IL 2, suggesting that CLL B cells could stimulate IL 2 receptor generation in normal T cells in an allo MLR, but not IL 2 production. The presence of IL 2 receptors on activated T cells was directly determined using anti- Tac, a monoclonal antibody with specificity for the IL 2 receptor. Of the mitogen- or MLR-activated T cells in CLL patients, 6% and 10%, respectively, expressed Tac antigen, whereas identically stimulated control T cells were 60% and 47% Tac+, respectively. Our findings suggest that T cells in B-CLL are defective in their recognition of self or foreign major histocompatibility antigens as demonstrated by their impaired responsiveness in the MLR. Thus, these cells are unable to produce IL 2 or generate IL 2 receptors.

Sphingosine 1-Phosphate (S1P) Induces Migration and ERK/MAP-Kinase-Dependent Proliferation in Chronic Lymphocytic Leukemia (B-CLL) Due to Expression of the G Protein-Coupled Receptors S1P1/4.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 4996-4996
Author(s):  
Gabriele Seitz ◽  
Sedat Yildirim ◽  
Andreas M. Boehmler ◽  
Lothar Kanz ◽  
Robert Möhle
Keyword(s):  
Chronic Lymphocytic Leukemia ◽  
G Protein ◽  
Cell Lines ◽  
Peripheral Blood ◽  
G Protein Coupled Receptors ◽  
Lymphocytic Leukemia ◽  
S1p Receptors ◽  
Sphingosine 1 Phosphate ◽  
G Protein Coupled

Abstract Egress of lymphocytes from lymphoid organs into the circulation has been shown to depend on the presence of the lipid mediator sphingosine 1-phosphate (S1P) in the peripheral blood, and expression of corresponding S1P receptors (i.e., S1P1), that belong to the family of 7-transmembrane G protein-coupled receptors (GPCR). As circulating lymphocytic lymphoma cells are a hallmark of chronic lymphocytic leukemia, we analyzed expression of different S1P receptors and the effects of S1P on B-CLL cells. By qualitative and quantitative (TaqMan) RT-PCR, significant mRNA expression of S1P1 and S1P4 was found in CLL cell lines (EHEB, MEC-1) and in most samples (S1P1 in 88%, S1P4 in 100%) of primary CD19+ cells isolated from the peripheral blood of untreated B-CLL patients. mRNA of other S1P receptors (S1P2, S1P3, S1P5) was less consistently detected. Normal, nonmalignant B cells were strongly positive for S1P1, while other S1P receptors were weakly expressed or negative. S1P induced typical effects of chemotactic GPCR, such as actin polymerization (analyzed by flow cytometry) and chemotaxis (measured in a modified Boyden chamber assay) in CLL cell lines and primary B-CLL cells. After serum deprivation in vitro, S1P induced phosphorylation of ERK/MAP-kinase as analyzed by Western blot, demonstrating that S1P receptors expressed in CLL were able to activate signaling pathways of GPCR not only related to cell migration and chemotaxis, but also to cell proliferation. Of note, the S1P1 ligand FTY720, which induces receptor internalization after prolonged exposure and acts as an antagonist, resulted in apoptosis in CLL cell lines and primary CLL cells in vitro, as measured by MTT-test and staining with Annexin-FITC, respectively. We conclude that sphingosine 1-phosphate, which is present in the peripheral blood in considerable amounts, contributes to the trafficking of B-CLL cells expressing the GPCRs S1P1/4, and to their prolonged survival.

Silvestrol, a Rocaglate Derivative from the Indonesian Plant Aglaia foveolata, Has Significant Bcl-2- and p53-Independent Anti-Tumor Activity against Chronic Lymphocytic Leukemia Cells.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 2600-2600 ◽  
Author(s):  
Ryan B. Edwards ◽  
David M. Lucas ◽  
Gerard Lozanski ◽  
Amy J. Johnson ◽  
Bao-Ning Su ◽  
...  
Keyword(s):  
T Cells ◽  
Chronic Lymphocytic Leukemia ◽  
Mtt Assay ◽  
Whole Blood ◽  
Mononuclear Cells ◽  
Lymphocytic Leukemia ◽  
Equal Distribution ◽  
Significant Difference ◽  
Anti Tumor Activity

Abstract Chronic Lymphocytic Leukemia (CLL) is an incurable disease with limited therapeutic options. The development of drug resistance through multiple pathways, especially in advanced disease, further restricts these options. Thus, new agents with unique mechanisms of action are crucial to make an impact on patient survival. Silvestrol, a rocaglate derivative with an unusual dioxanyloxy unit, was isolated from Aglaia species using bioassay-guided fractionation. Silvestrol exhibited potent in vitro cytotoxic activity against several tumor cell lines. Silvestrol was further evaluated in vivo in the hollow fiber test and in the murine P-388 leukemia model, in which it demonstrated promising anti-tumor activity with no significant weight loss up to 2.5 mg/kg (3.7 μM, assuming equal distribution) (1). Based on these results, we tested silvestrol against tumor cells obtained from CLL patients. Silvestrol exhibited significant antitumor activity with an estimated LC50 (concentration lethal to 50% of cells relative to untreated control) of 10 nM at 72 hours by MTT assay. In contrast, at this same timepoint using normal human peripheral blood mononuclear cells, an LC50 for silvestrol could not be defined even up to 4.0 μM. Under identical conditions, silvestrol was 50 to 100 fold more potent than the active metabolite of fludarabine, commonly used in the treatment of CLL. To determine the minimum exposure time required for silvestrol to have an effect, cells were incubated for various times in 80 nM silvestrol, then washed and resuspended in media with or without drug and incubated for a total of 72 hours. With only a four hour exposure, an average of 56% cytotoxicity was observed relative to untreated cells, and with a 24 hour exposure, results between samples in which the drug was removed and those incubated continuously were indistinguishable. T cell depletion and concomitant immunodeficiency is a serious risk with therapies currently available for CLL. We therefore tested the relative effects of silvestrol on B and T cells. By MTT assay with selected cells and in whole blood incubations followed by flow cytometry, using blood from both CLL patients and healthy volunteers, silvestrol demonstrated significantly more cytotoxicity toward B cells than T cells. Although some variability was observed between patient samples, silvestrol had activity against all samples tested and there was no detectable difference in average potency against cells from patients with a 17p13 deletion (chromosomal site of p53) relative to those without this risk factor. Furthermore, there was no significant difference in silvestrol-mediated cytotoxicity between lymphoblastic cells with a ten-fold overexpression of Bcl-2 relative to control cells. Together, these data demonstrate that silvestrol has efficacy against CLL cells in vitro and in whole blood, has highly unusual B-cell specificity, and is independent of key CLL resistance mechanisms. Our data strongly support further investigation of silvestrol as an antitumor agent in CLL. Studies are underway to determine the precise mechanism of action of this compound in CLL cells.

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