Targeting CK2 Overexpression as a Novel Therapeutic Tool in Chronic Lymphocytic Leukemia.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 803-803
Author(s):  
Leila R. Martins ◽  
Paulo Lúcio ◽  
Paula Gameiro ◽  
Maria Gomes Silva ◽  
Joao T Barata
Keyword(s):  
Chronic Lymphocytic Leukemia ◽  
Cell Survival ◽  
Peripheral Blood ◽  
Leukemia Cell ◽  
Lymphocytic Leukemia ◽  
Great Promise ◽  
Dependent Manner ◽  
Healthy Controls ◽  
Primary Leukemia

Abstract Abstract 803 Characterization of the molecular mechanisms that regulate viability of B cell chronic lymphocytic leukemia (CLL) cells may provide novel insights into the biology of this incurable malignancy and reveal prognostic markers and therapeutic targets. In particular, specific inhibition of signaling elements essential for leukemia cell survival offers great promise for the design of more efficient and selective therapies. The protein serine/threonine kinase CK2 is frequently upregulated in different cancers and mounting evidence implicates CK2 in tumorigenesis. In the present study, we evaluated whether CK2 may play a significant role in CLL. Peripheral blood mononuclear cells (PBMCs) from CLL patients (n=15) and healthy controls (n=7 ) were initially compared by Western blot densitometry analysis. CK2α, but not CK2β, was significantly upregulated in primary CLL patient samples (P=0.0003; Unpaired t-test), and CK2α expression clearly correlated with CK2 in vitro kinase activity (P value=0.0165). To test the functional significance of CK2 overexpression in CLL, we cultured PBMCs collected from leukemia patients (n=53) or purified CLL cells (n=7) in the presence of the CK2-specific small molecule inhibitors TBB and DRB. As determined by Annexin V-APC/7AAD staining and analysis of procaspase 3 cleavage, both TBB and DRB clearly promoted apoptosis of CLL cells (CD19+CD5+) in a time- and dose-dependent manner. Importantly, neither CK2 antagonist induced significant cell death in the normal T-cell population (CD19-CD3+) present in each PBMC patient sample. Consequently, the percentage of normal T-cells dramatically increased upon TBB/DRB treatment. Further, B-cells from healthy controls (n=7) were not affected by TBB or DRB, confirming the selectivity of the CK2 inhibitors towards leukemia cells. Notably, although co-culture with OP9 stromal cells promoted primary leukemia cell survival in vitro, it did not prevent apoptosis of CLL cells mediated by CK2 inhibition. The pro-apoptotic effect of TBB/DRB did not correlate with clinical parameters such as lymphocyte doubling time, ZAP-70 expression or IGHV mutational status. Interestingly, there was a significant association between the percentage of CLL cells in the peripheral blood and sensitivity to CK2 inhibition (e.g. 25μM TBB, P=0.0043), and treatment with 12.5μM TBB correlated with Binet stage (P=0.0043). We previously showed that CK2 phosphorylates and thereby inactivates PTEN in primary T-ALL cells leading to the hyperactivation of PI3K signaling pathway (Silva et al, JCI 2008). Here, we found that primary CLL samples displayed higher P-PTEN/PTEN ratios than healthy controls (P=0.0034), which correlated with CK2 expression (P=0.0064). Furthermore, treatment with TBB/DRB abrogated PTEN phosphorylation in 2 primary leukemia samples analyzed, suggesting that CK2 negatively regulates the activity of the tumor suppressor PTEN in CLL and raising the possibility that the pro-survival effect of CK2 may be dependent, at least in part, on the activation of PI3K pathway. Overall, our study suggests that CK2 plays an important role in the biology of CLL, and lays the groundwork for the inclusion of CK2 antagonists into future therapeutic options. Disclosures: No relevant conflicts of interest to declare.

Lenalidomide Abrogates the Protective Influence of Nurse-Like Cells on Primary Chronic Lymphocytic Leukemia Cells In Vitro.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 3116-3116 ◽  
Author(s):  
Danelle F. James ◽  
Maryann R. Betty ◽  
Ruzbeh Mosadeghi ◽  
Thomas J. Kipps
Keyword(s):  
Chronic Lymphocytic Leukemia ◽  
Cell Viability ◽  
Cell Survival ◽  
Leukemia Cell ◽  
Lymphocytic Leukemia ◽  
Response To Treatment ◽  
Leukemia Cells ◽  
High Dependency ◽  
Cells Cultured

Abstract Lenalidomide (3-(4-amino-1-oxo-3H-isoindol-2-yl)piperidine-2,6-dione)) is an agent approved for treatment of patients with del 5q myelodysplastic syndromes and previously treated multiple myeloma. Lenalidomide has been found in early clinical trials to have potential therapeutic activity in patients with relapsed chronic lymphocytic leukemia (CLL). The mechanism(s) whereby this drug is active in CLL is unknown. In particular, studies to date have not found lenalidomide to have any direct cytotoxic activity on CLL cells in vitro. This has stimulated speculation that this agent might adversely affect the positive influence of the microenvironment on leukemia-cell survival. We and others have observed that cells found in the leukemia microenvironment can support CLL-cell survival in vitro. One such type of cells are nurse-like cells (NLC), which can differentiate from the CD14-positive blood mononuclear cells of CLL patients into large, round adherent cells that can attract and support CLL cell survival in vitro for weeks, if not longer. We evaluated the effects of lenalidomide on primary leukemia-cell survival in vitro when the CLL cells from different patients (N=21) were cultured alone or together with NLC generated as previously described [Tsukada Blood 2002]. We assessed the in-vitro activity of lenalidomide on primary CLL cells from 21 patients, in duplicate in a series of 6 experiments. Lenalidomide at concentrations of 0.1μM-200μM did not significantly impact the survival of CLL cells that were cultured alone for up to 12 days. Analysis of cell surface markers revealed increased expression of CD38 at 36 hours in 5/5 lenalidomide treated CLL samples compared with untreated cells (MFIR 5.7 +/− .86 vs. 3.4 +/− .83 p=.003). We observed sustained upregualtion of CD40 and regulation of CXCR4 in the majority of cells treated with lenalidomide. When cultured with NLC, the survival of CLL cells was comparable to or significantly higher than that of CLL cells cultured alone 62.4% vs. 51% (+/−3% SEM n=21 p [<] 0.0005). The addition of lenalidomide at concentrations of 0.1μM and greater to co-cultures of NLC and CLL cells caused specific reductions in CLL cell survival to levels similar to or lower than that of CLL cells cultured without NLC. In the presence of NLC, lenalidomide at 1μM reduced CLL cell viability compared to control (41.5% vs. 56% +/−4% p [<] 0.0005 paired student t test n=13). For most patients the levels of CLL cell viability on days 4 through 8 in the co-cultures with lenalidomide was significantly lower than those of CLL cells co-cultured with NLC in the absence of lenalidomide. As such, this study reveals that physiologic concentrations of lenalidomide might abrogate the protective influence of NLC on CLL cell survival in vitro and potentially in vivo. Conceivably, those patients who have leukemia cells displaying a high dependency on NLC for survival in vitro also might be most likely to experience a favorable clinical response to treatment with lenalidomide. This hypothesis will be tested in a prospective manner with a planned clinical trial evaluating lenalidomide for treatment of CLL through the CLL Research Consortium.

Mode of Action of the SDF-1/CXCL12 Inhibiting Spiegelmer® Nox-A12 and Its Impact On Chronic Lymphocytic Leukemia (CLL) Cell Motility and Chemosensitization

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 318-318
Author(s):  
Dirk Zboralski ◽  
Julia Hoellenriegel ◽  
Christian Maasch ◽  
Anna Kruschinski ◽  
Jan A. Burger
Keyword(s):  
Bone Marrow ◽  
Chronic Lymphocytic Leukemia ◽  
Stromal Cells ◽  
Mode Of Action ◽  
Leukemia Cell ◽  
Lymphocytic Leukemia ◽  
Lymphoid Tissues ◽  
Cell Trafficking ◽  
Dependent Manner

Abstract Abstract 318 NOX-A12 is a novel Spiegelmer®-based antagonist of SDF-1/CXCL12, a chemokine involved in the regulation of chronic lymphocytic leukemia (CLL) cell trafficking. Spiegelmers® are mirror-image oligonucleotides that are identified to specifically bind to proteins in a manner conceptually similar to antibodies. Unlike aptamers, however, Spiegelmers® are built from the non-natural L-isomer form of nucleotides which confers resistance to the action of nucleases and avoids potential immunogenicity. CXCL12 is constitutively secreted and presented by bone marrow stromal cells (BMSC) via glycosaminoglycans (GAG) and acts as a homing factor for normal and malignant hematopoietic cells to the bone marrow (BM) and secondary lymphoid tissues via CXCR4 receptors that are expressed at high levels on circulating CLL cells. The microenvironment in the BM and secondary lymphoid tissues, in particular the CXCL12-CXCR4 axis, favors survival and chemotherapy-resistance of leukemic cells. We therefore investigated the effects of NOX-A12 in an in vitro co-culture system to model the interaction of CLL cells with their microenvironment. Surprisingly we observed that NOX-A12 increased pseudoemperipolesis in vitro, i.e. spontaneous leukemia cell migration beneath BMSC. Interestingly, this NOX-A12 induced trans-migration of CLL cells was completely inhibited by the CXCR4 antagonist AMD3100, suggesting a CXCL12/CXCR4 dependent mechanism. We postulated that this observation might result from a direct effect of NOX-A12 on CXCL12 release by the stromal cells. Therefore, we investigated this hypothesis in different BMSC lines (MS-5, R15C, and TSt-4) and we found that NOX-A12 induced a significant CXCL12 release in all three tested cell lines. We asked whether this NOX-A12 dependent increase of CXCL12 of BMSCs is due to release from either intracellular or extracellular storages. Intracellular staining of CXCL12 using flow cytometry did not reveal significant changes when BMSCs were incubated with NOX-A12. Furthermore, the transcription of CXCL12 was not found to be altered after NOX-A12 incubation over a period of three days as shown by quantitative RT-PCR. Rather, CXCL12 is released from extracellular storages of BMSCs. First hints were obtained through a rapid CXCL12 release within five minutes of incubation with NOX-A12. To confirm that CXCL12 is bound to the extracellular surface (by GAGs like heparin) and is being detached by NOX-A12 we first incubated BMSCs with NOX-A12, followed by a wash step and the addition of recombinant CXCL12. Recombinant CXCL12 was bound by BMSCs that were pre-incubated with NOX-A12 but not with a non-functional control (revNOX-A12), indicating that NOX-A12 strips off CXCL12. To corroborate the findings we incubated the BMSCs with heparin which also led to the release of CXCL12 in a dose dependent manner. Of note, the EC50 of heparin regarding CXCL12 release was much higher compared to the EC50 of NOX-A12 (≈ 12 μM vs. 5 nM) revealing the high affinity of NOX-A12 to CXCL12. The competition of NOX-A12 with heparin regarding CXCL12 binding was confirmed by Biacore experiments. Based on these findings, we developed a novel adapted co-culture approach to examine the ability of NOX-A12 to chemosensitize CLL cells. In this setting, we first strip off CXCL12 from BMSCs by NOX-A12 and subsequently add CLL cells which will be either non-treated or treated with chemotherapy (fludarabine combined with bendamustine). We found that NOX-A12 slightly decreased CLL cell viability. As expected, a strong viability decrease was observed with chemotherapy, which could be even further decreased by the combination with NOX-A12, suggesting synergistic effects. In conclusion, we propose that NOX-A12's mode of action is the release of extracellular bound CXCL12 and its subsequent inhibition. Since CXCL12 induces leukemia cell trafficking and homing to tissue microenvironment and also favors leukemia cell survival, we believe that targeting CXCL12 is an attractive approach to remove the protective effects of CXCL12-secreting BMSCs in order to sensitize CLL cells for subsequent chemotherapy. Thus, NOX-A12 represents a very promising agent to significantly improve the treatment of CLL. The compound is currently being tested in a Phase IIa study in relapsed CLL patients. Disclosures: Zboralski: NOXXON Pharma AG, Berlin, Germany: Employment. Maasch:NOXXON Pharma AG: Employment. Kruschinski:NOXXON Pharma AG: Employment. Burger:NOXXON Pharma AG: Consultancy, Research Funding.

In Vitro and in Vivo Targeting of Chronic Lymphocytic Leukemia Using CX-4945, a Clinical-Stage CK2-Specific Inhibitor.

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 2897-2897 ◽  
Author(s):  
Leila R. Martins ◽  
Paulo Lúcio ◽  
Alice Melao ◽  
Bruno A. Cardoso ◽  
Ryan Stansfield ◽  
...  
Keyword(s):  
Clinical Trials ◽  
Chronic Lymphocytic Leukemia ◽  
Cell Survival ◽  
Leukemia Cell ◽  
Lymphocytic Leukemia ◽  
Student’S T ◽  
The Impact ◽  
Student’S T Test

Abstract Abstract 2897 Specific inhibition of signaling elements essential for chronic lymphocytic leukemia (CLL) cell survival offers great promise for the design of improved therapies against this still incurable malignancy. The serine/threonine protein kinase CK2 is frequently upregulated in cancer, and mounting evidence implicates CK2 in tumorigenesis. Here, we evaluated whether CK2 is a valid target for therapeutic intervention in CLL, by testing the efficacy of CX-4945, a potent and highly specific orally available ATP-competitive inhibitor of CK2 that is undergoing phase I clinical trials for solid tumors and multiple myeloma. We previously showed that CK2 phosphorylates and thereby inactivates PTEN in primary T-ALL and CLL cells, leading to the hyperactivation of PI3K signaling pathway, and consequently promoting leukemia cell survival (Silva et al, JCI 2008; Martins et al, Blood 2010). Therefore, we first analyzed the impact of CX-4945 on PTEN phosphorylation and PI3K pathway activation. Incubation of CLL cells with 20 μM CX-4945 for 2h resulted in striking downregulation of PTEN phosphorylation, indicative of increased PTEN activity, and a concomitant decrease in the activity of PI3K downstream targets Akt and PKC, as determined by Akt (S473), PKCβ (S660) and PKCδ (T550) phosphorylation in both MO1043 and primary CLL cells collected and isolated to >90% purity from the peripheral blood of untreated patients. Importantly, we confirmed that Akt phosphorylation on the CK2 direct target site (S129) was also inhibited by CX-4945. Next, we evaluated the functional impact of the CK2 inhibitor on CLL cell viability. Primary CLL cells (n=11) were cultured with 10 and 20 μM CX-4945. Both drug concentrations exerted clear pro-apoptotic effects in all cases (P<0.0001 for each dose, 2-tailed paired Student's t test), as determined by Annexin V-APC/7-AAD staining. Moreover, the effect of CX-4945 was time- and dose-dependent in 4 out of 4 cases that were more thoroughly analyzed. Similar results were obtained using MEC1, MEC2, WaC3CD5, JVM3 and MO1043 cell lines whose IC50 ranged between 3.1 and 5.8μM. Notably, although co-culture with OP9 stromal cells promoted primary leukemia cell survival, it did not prevent CX-4945-mediated apoptosis of CLL cells. Most importantly, CX-4945 induced a stronger decrease in the viability of CLL cells from patients with higher percentage of malignant cells in the blood (R2=0.4176, P=0.0317, n=11, Pearson correlation), Binet stage B/C (P=0.0424, n=10, 2-tailed unpaired Student's t test) or higher plasma β2 microglobulin levels (P=0.0239, n=9). Furthermore, CLL cells with a higher proliferation rate (LDT < 12 months) were also more sensitive to CX-4945 (P=0.0007, n=11). In accordance, the need for treatment positively correlated with the sensitivity to CX-4945 (R2=0.4504, P = 0.0238). These observations suggest that treatment with CK2 inhibitors may be especially beneficial to patients with more advanced or aggressive disease. The promising results obtained in vitro prompted us to assess the impact of CX-4945 on CLL tumor development in vivo. We implanted MO1043 CLL cells subcutaneously into Swiss nude mice. At day 3, all animals presented palpable tumor masses of approximately 150 mm3, and were randomly assigned into 4 groups (n=6 per group) to receive either CX-4945 alone (75mg/kg, bid, p.o.), fludarabine alone (34mg/kg, i.p., 5 days + 2 days rest, every week), the combination of both drugs, or vehicle control. A significant delay in tumor growth was observed in all of the treatment groups when compared to the control group (P<0.0001, 2-way ANOVA). Notably, CX-4945 was as effective as fludarabine when used as a single agent, and the combination of the two drugs was significantly more effective than fludarabine alone (P=0.0375). All treatments were well tolerated as evidenced by the maintenance of body weight and the inexistence of signs of overt toxicity. Overall, our data indicate that pharmacological inhibition of CK2 is a promising therapeutic strategy in CLL that may be of special benefit to patients with aggressive and advanced stage disease. Moreover, our studies pave the way to the development of clinical trials using CX-4945 or other CK2 antagonists to manage CLL. Disclosures: Stansfield: Cylene Pharmaceuticals Inc.: Employment. Drygin:Cylene Pharmaceuticals Inc.: Employment.

Chronic Lymphocytic Leukemia Cells Can Induce Monocytes to Express High Levels of BAFF and Assume Properties of Nurselike Cells In Vitro.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 588-588
Author(s):  
Davorka Messmer ◽  
Tomoyuki Endo ◽  
Bradley T. Messmer ◽  
Thomas J. Kipps
Keyword(s):  
Gene Expression ◽  
Flow Cytometry ◽  
Chronic Lymphocytic Leukemia ◽  
Cell Survival ◽  
Mononuclear Cells ◽  
Leukemia Cell ◽  
Lymphocytic Leukemia ◽  
Soluble Factor ◽  
Blood Mononuclear Cells

Abstract CD14+ blood mononuclear cells co-cultured with chronic lymphocytic leukemia (CLL) B cells differentiate into nurselike cells (NLCs) that in turn can support CLL-cell survival in vitro and possibly in vivo. These cells appear similar to lymphoma-associated macrophages, which were identified in secondary lymphoid tissue of patients with follicular lymphoma and appear more prevalent in patients with therapy-resistant disease. To investigate the relationship between NLC and macrophages, we performed studies on macrophages and NLCs that were induced to differentiate from CD14+ blood mononuclear cells in vitro. Consistent with prior studies, we found that NLCs express significantly higher levels of CD68 than macrophages, as assessed via cytoplasmic flow cytometry. However, Affymatrix U113A microarray analysis of gene expression by NLC, macrophages, and monocytes-derived dendritic cells (DCs) from 3 donors revealed major differences in gene expression between DCs versus macrophages or NLCs, but no major differences in gene expression profiles between NLCs and macrophages. Flow cytometric analyses of NLCs and macrophages revealed that these two cell types also shared similar expression levels of CD16, CD32, CD35, CD86, CD58, MHC-II, CD40, and CD54. However, using flow cytometry we found that NLCs (n=9) expressed significantly higher levels than macrophages of the B-cell activating factor belonging to the tumor necrosis factor family (BAFF). Deconvolution microscopy confirmed the differences in BAFF expression and also revealed that NLCs express higher levels of a proliferation-inducing ligand (APRIL) than macrophages. These are two key factors involved in promoting leukemia/lymphoma B cell survival. Moreover, NLCs maintained high-level expression of BAFF even when cultured apart from CLL cells in fresh medium. We investigated whether co-culture of differentiated macrophages with CLL cells could induce the macrophages to express high-levels of BAFF. Although such co-culture induced progressive increase in macrophages-expression of BAFF, the levels of BAFF induced after even 7 days of co-culture were lower than those noted for NLCs. We cultured CD14+ blood monocytes and CLL cells separated across a transwell membrane to determine whether a soluble factor(s) was responsible for the induction of high BAFF levels noted on NLCs. Following several days in culture, the cultured monocytes acquired expression levels of BAFF similar to those detected for NLCs. These studies indicate that monocytes can respond to a soluble factor(s) elaborated by CLL cells to assume properties similar to those of NLCs. Moreover they suggest that NLCs may be a peculiar type of terminally differentiated macrophages-like cells induced by the leukemia-cell population to have properties that promote CLL cell survival. Agents that can block the maturation of monocytes into NLCs or that inhibit the capacity of NLCs to promote leukemia-cell survival may be effective in the treatment of CLL and related lymphoid malignancies.

scholarly journals Chlorambucil effect on B lymphocites in the peripheral blood of patients with chronic lymphocytic leukemia: Cell ultrastructure investigation

2003 ◽  
Vol 60 (2) ◽  
pp. 175-180 ◽  
Author(s):  
Goran Brajuskovic ◽  
Slobodan Marjanovic ◽  
Andjelija Skaro-Milic
Keyword(s):  
Chronic Lymphocytic Leukemia ◽  
Peripheral Blood ◽  
Antineoplastic Agent ◽  
Experimental Studies ◽  
Leukemia Cell ◽  
Cell Ultrastructure ◽  
Lymphocytic Leukemia ◽  
Chronic Lymphocytic Leukemia Cell

B type Chronic Lymphocytic Leukemia (B-CLL) is a malignant disease characterized by the progressive accumulation of morphologically mature, but immunologically dysphunctional CD 5+ lymphocytes in the blood, bone marrow and lymphatic organs in the early phase of the cell cycle. B-CLL is an example of human malignancy caused by alternations in the pathways of programmed cell death - apoptosis. Recent investigations showed a probable role of apoptosis as a prognostic parameter in B-CLL patients. Since the introduction of chlorambucil in the therapy in 1952, besides all the achievements in modern oncology, chlorambucil remained the most common antineoplastic agent in the treatment of CLL. Numerous experimental studies both in vitro and in vivo, showed the capability of antineoplastic agents to induce the process of apoptosis of neoplastically transformed cells. In this study the effect of chlorambucil on B lymphocites was monitored in 16 samples of peripheral blood tarlen from B-CLL diagnosed patients. According to the investigations performed in this study by ultrastructure analysis of B-CLL cells, it was concluded that chlorambucil either induced apoptosis in B-CLL cells, or activated cell response to the stress.

BAFF Support Survival of Chronic Lymphocytic Leukemia B Cells by a Pathway Independent of Stromal Derived Factor-1α.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 1908-1908
Author(s):  
Mitsufumi Nishio ◽  
Nobuhiro Tsukada ◽  
Shinichi Kitada ◽  
Junko Ohata ◽  
Nathan J. Zvaifler ◽  
...  
Keyword(s):  
B Cells ◽  
Chronic Lymphocytic Leukemia ◽  
Cell Survival ◽  
Mononuclear Cells ◽  
Leukemia Cell ◽  
Lymphocytic Leukemia ◽  
Mitogen Activated Protein Kinase ◽  
Peripheral Blood Mononuclear ◽  
Cells Cultured

Abstract We examined the peripheral blood mononuclear cells (PBMC) of patients with chronic lymphocytic leukemia (CLL) for expression of B cell-activating factor of the TNF family (BAFF). Isolated CLL B cells had significantly lower levels of BAFF mRNA than did non-separated PBMC. Most of the BAFF mRNA in PBMC was due to contaminating CD14+ cells that previous studies found could differentiate into “nurselike” cells (NLC) when cultured with CLL B cells in vitro. We found NLC expressed high-levels of BAFF and stromal cell-derived factor-1 alpha (SDF-1α), in contrast to CLL B cells. CLL B cells cultured with exogenous recombinant human BAFF (rhBAFF), SDF-1α, or NLC sustained significantly greater viability than isolated CLL B cells cultured alone. The effect(s) of rhBAFF on leukemia cell survival appeared additive and distinct from that of SDF-1α, which in contrast to rhBAFF induced leukemia-cell phosphorylation of p44/42 mitogen-activated protein-kinase (ERK 1/2) and phosphorylation and activation of AKT at Ser473. However, rhBAFF, but not SDF-1α, could induce processing of p100 NF-κB2 to p52 and, like NLC, enhance and/or maintain CLL-cell expression of the anti-apoptotic protein Mcl-1. We conclude that BAFF can function in a paracrine manner to support leukemia cell survival via mechanisms that are distinct from those of SDF-1α and that NLC use multiple distinct pathways to support CLL-cell survival.

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 Involvement of CED-3/ICE Proteases in the Apoptosis of B-Chronic Lymphocytic Leukemia Cells

Blood ◽  
1997 ◽  
Vol 89 (9) ◽  
pp. 3378-3384 ◽  
Author(s):  
Beatriz Bellosillo ◽  
Mireia Dalmau ◽  
Dolors Colomer ◽  
Joan Gil
Keyword(s):  
Chronic Lymphocytic Leukemia ◽  
Lymphocytic Leukemia ◽  
Interleukin 4 ◽  
Leukemia Cells ◽  
Parp Cleavage ◽  
Dependent Manner ◽  
Kinase C ◽  
Dose Dependent ◽  
Pkc Activation

Abstract B-chronic lymphocytic leukemia (B-CLL) is characterized by the accumulation of long-lived B lymphocytes that express high levels of Bcl-2. We examined the involvement of CED-3/ICE-like proteases in the apoptosis of B-CLL cells. One of the substrates of these proteases is poly(ADP [adenosine 5′-diphosphate]-ribose) polymerase (PARP). The effect of different factors that induce the apoptosis of B-CLL cells on the proteolytic cleavage of PARP has been studied. Treatment of B-CLL cells with different concentrations of dexamethasone (1 to 1,000 μmol/L) induced in a dose-dependent manner the cleavage of PARP. Dexamethasone induced PARP cleavage after 12 hours of incubation, which was almost complete at 48 hours. PARP cleavage during apoptosis of B-CLL cells was studied in cells from eight patients and a correlation was found between cell viability and the degree of PARP cleavage. Incubation in vitro of B-CLL cells with fludarabine for 48 hours induced PARP cleavage in all the cases studied. Protein kinase C (PKC) activation with 100 nmol/L TPA (12-O-tetradecanoylphorbol 13-acetate) or incubation with interleukin-4 (10 ng/mL) prevented either dexamethasone- or fludarabine-induced proteolysis of PARP. Incubation of B-CLL cells with the CED-3/ICE–like protease inhibitor Z-VAD.fmk inhibited spontaneous and dexamethasone-induced PARP cleavage and DNA fragmentation in a dose-dependent manner. Furthermore, Z-VAD.fmk prevented the cytotoxic effect of dexamethasone. These results indicate that CED-3/ICE–like proteases play an important role in the apoptosis of B-CLL cells.

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.

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