Functional integrity of the p53-mediated apoptotic pathway induced by the nongenotoxic agent nutlin-3 in B-cell chronic lymphocytic leukemia (B-CLL)

Blood ◽  
2006 ◽  
Vol 107 (10) ◽  
pp. 4122-4129 ◽  
Author(s):  
Paola Secchiero ◽  
Elisa Barbarotto ◽  
Mario Tiribelli ◽  
Carlotta Zerbinati ◽  
Maria Grazia di Iasio ◽  
...  
Keyword(s):  
Chronic Lymphocytic Leukemia ◽  
B Cell ◽  
Mononuclear Cells ◽  
Lymphocytic Leukemia ◽  
Mrna Levels ◽  
Apoptotic Pathway ◽  
Mitochondrial Potential ◽  
Peripheral Blood Mononuclear ◽  
P53 Pathway ◽  
Cell Chronic Lymphocytic Leukemia

Deletions and/or mutations of p53 are relatively rare and late events in the natural history of B-cell chronic lymphocytic leukemia (B-CLL). However, it is unknown whether p53 signaling is functional in B-CLL and if targeted nongenotoxic activation of the p53 pathway by using nutlin-3, a small molecule inhibitor of the p53/MDM2 interaction, is sufficient to kill B-CLL cells. In vitro treatment with nutlin-3 induced a significant cytotoxicity on primary CD19+ B-CLL cells, but not on normal CD19+ B lymphocytes, peripheral-blood mononuclear cells, or bone marrow hematopoietic progenitors. Among 29 B-CLL samples examined, only one was resistant to nutlin-3–mediated cytotoxicity. The induction of p53 by nutlin-3 in B-CLL samples was accompanied by alterations of the mitochondrial potential and activation of the caspase-dependent apoptotic pathway. Among several genes related to the p53 pathway, nutlin-3 up-regulated the steady-state mRNA levels of PCNA, CDKN1A/p21, GDF15, TNFRSF10B/TRAIL-R2, TP53I3/PIG3, and GADD45. This profile of gene activation showed a partial overlapping with that induced by the genotoxic drug fludarabine. Moreover, nutlin-3 synergized with both fludarabine and chlorambucil in inducing B-CLL apoptosis. Our data strongly suggest that nutlin-3 should be further investigated for clinical applications in the treatment of B-CLL.

The Receptor for Hyaluronic Acid Mediated Motility (RHAMM/CD168) Is a Potential Target for Immunotherapy of Patients with B-Cell Chronic Lymphocytic Leukemia.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 53-53 ◽  
Author(s):  
Krzysztof Giannopoulos ◽  
Iwona Hus ◽  
Li Li ◽  
Agnieszka Bojarska-Junak ◽  
Jochen Greiner ◽  
...  
Keyword(s):  
T Cells ◽  
Chronic Lymphocytic Leukemia ◽  
B Cell ◽  
Mononuclear Cells ◽  
Tissue Expression ◽  
T Cell Response ◽  
Lymphocytic Leukemia ◽  
Leukemic Cells ◽  
Peripheral Blood Mononuclear ◽  
Cell Chronic Lymphocytic Leukemia

Abstract Definition of appropriate target antigens might open new avenues to antigen targeted immunotherapies for patients with B-cell chronic lymphocytic leukemia (B-CLL). We screened the mRNA expression of tumor associated antigens (TAAs), from the literature (fibromodulin, survivin, OFA-iLRP, BAGE, G250, MAGE1, PRAME, proteinase, Syntaxin, hTERT, WT-1), and TAAs defined earlier by serological analysis of cDNA expression libraries from leukemic cells (PINCH, HSJ2, MAZ, MPP11, RHAMM/CD168, NY-Ren60). Peripheral blood mononuclear cells from 43 B-CLL patients and 20 healthy volunteers (HVs) were examined by conventional and quantitative RT-PCR. mRNA of RHAMM/CD168, fibromodulin, syntaxin and NY-Ren60 was expressed in 55–90%, mRNA of HSJ2, MAZ and OFA-iLRP in 90–100% of the patients. No expression of WT-1, h-TERT, BAGE, G250, MAGE1 and survivin was observed. Low (2–20%) expression frequencies of MPP11, PINCH, PRAME and proteinase were detected. RHAMM/CD168, fibromodulin, PRAME and MPP11 showed expression in B-CLL patients, but not in HVs. Because of the exquisite tissue expression of RHAMM/CD168 and its high expression frequency in B-CLL patients even in early stages of disease, mixed lymphocyte peptide culture (MLPC), enzyme-linked immunosorbent spot (ELISPOT) and flow cytometry were performed for antigen specific T cells. In MLPC, RHAMM specific responses by CD8+HLA-A2/R3tetramer+CCR7-CD45RAhigh effector T cells were detected. Moreover, we observed an enhanced RHAMM/CD168 specific CD8+ T cell response after vaccination in one from four HLA-A2 positive B-CLL patients vaccinated with tumor cell lysate pulsed dendritic cells. Therefore, RHAMM/CD168 is an interesting candidate antigen for future immunotherapies in both ZAP-70 (+) and ZAP-70 (−) B-CLL patients.

scholarly journals Interleukin 2 production in B cell chronic lymphocytic leukemia

Blood ◽  
1985 ◽  
Vol 66 (4) ◽  
pp. 840-847 ◽  
Author(s):  
JF Rossi ◽  
B Klein ◽  
T Commes ◽  
M Jourdan
Keyword(s):  
T Cells ◽  
Chronic Lymphocytic Leukemia ◽  
B Cell ◽  
Mononuclear Cells ◽  
Interleukin 2 ◽  
Lymphocytic Leukemia ◽  
Leukemic Cells ◽  
Peripheral Blood Mononuclear ◽  
Healthy Donors ◽  
Cell Chronic Lymphocytic Leukemia

Abstract Interleukin 2 (IL 2) production by phytohemagglutinin (PHA)-stimulated peripheral blood mononuclear cells (PBMCs) was investigated in 22 patients with active untreated B cell chronic lymphocytic leukemia (B- CLL) and in 15 healthy donors. PBMCs from healthy donors demonstrated an IL 2 synthesis of 12.4 +/- 10 U/mL. B-CLL PBMCs produced a significant amount of IL 2 (8 +/- 6.6 U/mL) despite the low percentage of T cells (13% +/- 8%) associated with this disease compared with that found in healthy donors (63% +/- 7.5%). If IL 2 production is expressed as units per milliliter per 10(4) T cells, its level in patients with B- CLL (1.1 U/mL/10(4) T cells) is five times greater than that of the controls (0.19 units). When expressed as units per milliliter per liter of blood, the B-CLL patients produce approximately 12 times as much IL 2 as controls. IL 2 production in normal controls was doubled after irradiation of PBMCs or addition of indomethacin. This increase was not seen with B-CLL PBMCs suggesting that the latter have been devoid of prostaglandin-producing normal IL 2 suppressor cells. By mixing normal or B-CLL T cells with non-T cells we found that T cells from patients with B-CLL stimulated by normal accessory cells produced the same amount of IL 2 as normal T cells. Moreover, B-CLL non-T cells (mainly B leukemic cells) produced no IL 2 themselves but played a much more efficient role in IL 2 production than did non-T cells from healthy donors. This was not due to detectable IL 1 production by these cells. The IL 2 produced by B-CLL PBMCs was partially purified and recovered in a 16,000 mol wt fraction, the same mol wt as IL 2 from normal cells.

Interleukin 2 production in B cell chronic lymphocytic leukemia

Blood ◽  
1985 ◽  
Vol 66 (4) ◽  
pp. 840-847
Author(s):  
JF Rossi ◽  
B Klein ◽  
T Commes ◽  
M Jourdan
Keyword(s):  
T Cells ◽  
Chronic Lymphocytic Leukemia ◽  
B Cell ◽  
Mononuclear Cells ◽  
Interleukin 2 ◽  
Lymphocytic Leukemia ◽  
Leukemic Cells ◽  
Peripheral Blood Mononuclear ◽  
Healthy Donors ◽  
Cell Chronic Lymphocytic Leukemia

Interleukin 2 (IL 2) production by phytohemagglutinin (PHA)-stimulated peripheral blood mononuclear cells (PBMCs) was investigated in 22 patients with active untreated B cell chronic lymphocytic leukemia (B- CLL) and in 15 healthy donors. PBMCs from healthy donors demonstrated an IL 2 synthesis of 12.4 +/- 10 U/mL. B-CLL PBMCs produced a significant amount of IL 2 (8 +/- 6.6 U/mL) despite the low percentage of T cells (13% +/- 8%) associated with this disease compared with that found in healthy donors (63% +/- 7.5%). If IL 2 production is expressed as units per milliliter per 10(4) T cells, its level in patients with B- CLL (1.1 U/mL/10(4) T cells) is five times greater than that of the controls (0.19 units). When expressed as units per milliliter per liter of blood, the B-CLL patients produce approximately 12 times as much IL 2 as controls. IL 2 production in normal controls was doubled after irradiation of PBMCs or addition of indomethacin. This increase was not seen with B-CLL PBMCs suggesting that the latter have been devoid of prostaglandin-producing normal IL 2 suppressor cells. By mixing normal or B-CLL T cells with non-T cells we found that T cells from patients with B-CLL stimulated by normal accessory cells produced the same amount of IL 2 as normal T cells. Moreover, B-CLL non-T cells (mainly B leukemic cells) produced no IL 2 themselves but played a much more efficient role in IL 2 production than did non-T cells from healthy donors. This was not due to detectable IL 1 production by these cells. The IL 2 produced by B-CLL PBMCs was partially purified and recovered in a 16,000 mol wt fraction, the same mol wt as IL 2 from normal cells.

scholarly journals Evidence for progenitors of chronic lymphocytic leukemia B cells that undergo intraclonal differentiation and diversification

Blood ◽  
1996 ◽  
Vol 87 (4) ◽  
pp. 1586-1594 ◽  
Author(s):  
M Dono ◽  
S Hashimoto ◽  
F Fais ◽  
V Trejo ◽  
SL Allen ◽  
...  
Keyword(s):  
B Cells ◽  
Chronic Lymphocytic Leukemia ◽  
B Cell ◽  
Dna Sequences ◽  
Mononuclear Cells ◽  
Lymphocytic Leukemia ◽  
Specific Primers ◽  
Region Gene ◽  
Peripheral Blood Mononuclear ◽  
Ancestral Gene

Peripheral blood mononuclear cells from five patients with IgG+ B-type chronic lymphocytic leukemia (B-CLL) were analyzed for the presence of clone-specific Ig H chain variable region gene mRNA transcripts linked to C mu and/or C alpha. This was assessed by (1) comparing the lengths of portions of the VHDJH of the IgG+ CLL clones with those of the mu and alpha isotype-expressing B cells, (2) performing clone-specific endonuclease digestion studies, and (3) determining the DNA sequences of the mu and alpha isotype-expressing cDNA. Thus, when B-cell mRNA from these five patients were reverse transcribed with C gamma-specific primers and then amplified by polymerase chain reaction, dominant cDNA were found with lengths corresponding to those of the IgG+ CLL B cell. In addition, in four cases, cDNA of lengths identical to those of the CLL B cell were detected when mRNA was reverse transcribed and amplified using c mu- and/or C alpha-specific primers, strongly suggesting clonal relatedness. These CLL-related mu- and alpha- expressing cDNA were present in greater amounts that unrelated (non- CLL) mu- and alpha-expressing cDNA from normal B cells that used genes of the same VH family. When the sequences of these CLL-related C mu- and C alpha-expressing cDNA were compared with those of the IgG+ CLL clones, it was clear that they were derived from the same ancestral gene as the IgG-expressing CLL B cell, thus documenting their common origin. Finally, nucleotide point mutations were observed in the mu- and alpha-expressing cDNA of certain patients, indicating divergence with the CLL. These data suggest that IgM+ B cells, which are precursors of the leukemic B cells, exist in increased numbers in the blood of most patients with IgG+ B-CELL and that these cells may differentiate, accumulate V genes mutations, and undergo isotype switching in vivo. In addition, the data are consistent with a sequential-hit model for the evolution of CLL.

RHAMM/CD168 Is a Novel Leukemia Associated Antigen with Prognostic Value for Patients with B-Cell Chronic Lymphocytic Leukemia.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 2773-2773
Author(s):  
Krzysztof Giannopoulos ◽  
Alexander Krober ◽  
Anna Dmoszynska ◽  
Jacek Rolinski ◽  
Hartmut Dohner ◽  
...  
Keyword(s):  
T Cells ◽  
Chronic Lymphocytic Leukemia ◽  
B Cell ◽  
Cd8 T Cells ◽  
Mononuclear Cells ◽  
Surrogate Marker ◽  
Lymphocytic Leukemia ◽  
Leukemic Cells ◽  
Free Survival ◽  
Cell Chronic Lymphocytic Leukemia

Abstract Background and Aims: Differential expression of molecules in patients with B-cell chronic lymphocytic leukemia (B-CLL) might define suitable targets for T cell based vaccines and/or antibody approaches. Methods: We assessed the mRNA expression of the tumor associated antigen (TAA) RHAMM/CD168 defined earlier by serological analysis of cDNA expression libraries (SEREX) from leukemic cells. Results: Peripheral blood mononuclear cells from 40 B-CLL patients and 20 healthy volunteers (HVs) were examined by quantitative RT-PCR. A leukemia-restricted expression of the antigen RHAMM/CD168 was observed in 39/40 B-CLL patients in contrast to the absence of its expression in HVs. To evaluate the immunogenicity of this novel LAA, mixed lymphocyte peptide cultures (MLPCs), followed by enzyme-linked immunosorbent spot (ELISPOT) and flow cytometry assays were performed to detect antigen-specific CD8+ T cells. RHAMM/CD168 specific responses by CD8+ HLA-A2/R3tetramer+CCR7-CD45RAhigh effector T cells were detected. As these CD8+ T cells contribute to the elimination of RHAMM+ CLL cells, we questioned whether expression of the antigen would be associated with a better survival. RHAMM/CD168 expression revealed to be higher in patients with unmutated IgVH status. RHAMM normalized against the housekeeping gene TATA binding protein (TBP), i.e. the RHAMM/TBP ratio was defined as a prognostic surrogate marker for B-CLL. B-CLL patients with a RHAMM/TBP ratio > 1.67 showed a significantly shorter treatment free survival (TFS) (Fig. 1). A tendency towards higher RHAMM/TBP expression ratios was observed in B-CLL cases with del11q. Conclusion: RHAMM/CD168 is a novel LAA in B-CLL patients, an antigen correlating with the clinical course of the disease. Therefore, we consider RHAMM/CD168 an interesting target for immunotherapy in early stage B-CLL patients, especially with worse prognosis (IgVH unmutated). Figure 1. Treatment-free-survival (TFS) according to RHAMM/TBP ratio Figure 1. Treatment-free-survival (TFS) according to RHAMM/TBP ratio

Tyro3, Axl Ve Mer (TAM) Receptors On Surfaces of Mononuclear Cells in Patients with B-Cell Chronic Lymphocytic Leukemia

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 4588-4588
Author(s):  
Dilvin Guney ◽  
Aysin Tulunay ◽  
Funda Pepedil ◽  
Isik Kaygusuz ◽  
Cafer Adiguzel ◽  
...  
Keyword(s):  
Chronic Lymphocytic Leukemia ◽  
B Cell ◽  
Mononuclear Cells ◽  
Conflicts Of Interest ◽  
Gradient Centrifugation ◽  
Lymphocytic Leukemia ◽  
Control Group ◽  
Ligand Molecule ◽  
Tam Receptors ◽  
Cell Chronic Lymphocytic Leukemia

Abstract Abstract 4588 Background: Tyro 3 (Sky), Axl, and Mer receptors are members of the family of tyrosine kinases and Gas6 is their ligand molecule. In some types of cancer, upregulation of Axl/Gas6 indicated a worse prognosis, but an opposite situation was observed in renal “cell” carcinoma. This contradiction may suggest that Axl/Gas6 pathway varies depending on the type of cancer. The objective of this study is to investigate TAM receptors on surfaces of mononuclear cells in patients with B-Cell chronic lymphocytic leukemia (B-Cell-CLL). Material & Methods: B-Cell-CLL patients (grade 0–1, according to the classification of RAI), who were not on a drug treatment, were recruited in this study (n= 20; 9 female, 11 male). Their ages were 44 to 74 (mean: 63), and the control group consisted of 13 healthy volunteers (5 female, 8 male), whose age range is 20–89 (mean: 36). Mononuclear cells were isolated by density gradient centrifugation, and then surface TAM receptors were detected by flow cytometry. Mononuclear cell were stained with the primary antibodies against Tyro3, Axl and Mer. Results: The percentage of the surface TAM receptors on mononuclear cells from the patient group (25–75% interquartile range): Tyro 3= 25.50 (4.2– 45.62); Axl= 17/55 (5.57– 36.32), and Mer= 19.90 (1.92– 37.55). In the control group the following values were obtained: Tyro 3= 2.60 (1.35–3.25); Axl= 0.9 (0.4–2.6), and Mer= 2.50 (0.35–3.65). The percentage of three of them was significantly higher in the B-Cell-CLL group than those in the control group (P<0.01). Conclusion: In conclusion, this preliminary study showed that TAM receptors on surfaces of mononuclear cells are higher in patients with B-Cell-CLL patients than the control group. Gas6/TAM signaling may play a potential role in the pathogenesis of B Cell-CLL. Further studies are required to elucidate the actual role of Gas6/TAM signaling in B-Cell-CLL. Gas6/TAM signaling might be a new strategic goal for the treatment of B-Cell-CLL. Disclosures: No relevant conflicts of interest to declare.

Survivin is expressed on CD40 stimulation and interfaces proliferation and apoptosis in B-cell chronic lymphocytic leukemia

Blood ◽  
2001 ◽  
Vol 97 (9) ◽  
pp. 2777-2783 ◽  
Author(s):  
Luisa Granziero ◽  
Paolo Ghia ◽  
Paola Circosta ◽  
Daniela Gottardi ◽  
Giuliana Strola ◽  
...  
Keyword(s):  
B Cells ◽  
Chronic Lymphocytic Leukemia ◽  
B Cell ◽  
Molecular Mechanisms ◽  
Mononuclear Cells ◽  
Survivin Expression ◽  
Lymphocytic Leukemia ◽  
Cd40 Stimulation ◽  
Cell Chronic Lymphocytic Leukemia

Abstract In B-cell chronic lymphocytic leukemia (B-CLL), defective apoptosis causes the accumulation of mature CD5+ B cells in lymphoid organs, bone marrow (BM), and peripheral blood (PB). These cells are the progeny of a proliferating pool that feeds the accumulating compartment. The authors sought to determine which molecular mechanisms govern the proliferating pool, how they relate to apoptosis, and what the role is of the microenvironment. To begin to resolve these problems, the expression and modulation of the family of inhibitor of apoptosis proteins (IAPs) were investigated, with consideration given to the possibility that physiological stimuli, such as CD40 ligand (CD40L), available to B cells in the microenvironment, might modulate IAP expression. The in vitro data on mononuclear cells from PB or BM of 30 patients demonstrate that B-CLL cells on CD40 stimulation express Survivin and that Survivin is the only IAP whose expression is induced by CD40L. Through immunohistochemistry, in vivo Survivin expression in lymph node (LN) and BM biopsies was evaluated. In reactive LN, Survivin was detected only in highly proliferating germinal center cells. In LN from patients with B-CLL, Survivin was detected only in pseudofollicles. Pseudofollicle Survivin+ cells were actively proliferating and, in contrast to Survivin+ B cells found in normal GC, were Bcl-2+. In B-CLL BM biopsies, CD5+, Survivin+ cells were observed in clusters interspersed with T cells. These findings establish that Survivin controls the B-CLL proliferative pool interfacing apoptosis and that its expression may be modulated by microenvironmental stimuli.

The natural product honokiol induces caspase-dependent apoptosis in B-cell chronic lymphocytic leukemia (B-CLL) cells

Blood ◽  
2005 ◽  
Vol 106 (2) ◽  
pp. 690-697 ◽  
Author(s):  
Traci E. Battle ◽  
Jack Arbiser ◽  
David A. Frank
Keyword(s):  
Chronic Lymphocytic Leukemia ◽  
B Cell ◽  
Natural Product ◽  
Mononuclear Cells ◽  
Single Agent ◽  
Lymphocytic Leukemia ◽  
Interleukin 4 ◽  
Response To Treatment ◽  
Potent Inducer ◽  
Cell Chronic Lymphocytic Leukemia

Abstract B-cell chronic lymphocytic leukemia (B-CLL) remains an incurable disease that requires innovative new approaches to improve therapeutic outcome. Honokiol is a natural product known to possess potent antineoplastic and antiangiogenic properties. We examined whether honokiol can overcome apoptotic resistance in primary tumor cells derived from B-CLL patients. Honokiol induced caspase-dependent cell death in all of the B-CLL cells examined and was more toxic toward B-CLL cells than to normal mononuclear cells, suggesting greater susceptibility of the malignant cells. Honokiol-induced apoptosis was characterized by the activation of caspase-3, -8, and -9 and cleavage of poly(adenosine diphosphate-ribose) polymerase (PARP). Exposure of B-CLL cells to honokiol resulted in up-regulation of Bcl2-associated protein (Bax) and down-regulation of the expression of the key survival protein myeloid-cell leukemia sequence 1 (Mcl-1), which is associated with response to treatment in B-CLL patients. In addition, B-CLL cells pretreated with interleukin-4 (IL-4), a cytokine known to support B-CLL survival, underwent apoptosis when subsequently incubated with honokiol, indicating that honokiol could also overcome the prosurvival effects of IL-4. Furthermore, honokiol enhanced cytotoxicity induced by fludarabine, cladribine, or chlorambucil. These data indicate that honokiol is a potent inducer of apoptosis in B-CLL cells and should be examined for further clinical application either as a single agent or in combination with other anticancer agents. (Blood. 2005;106:690-697)

scholarly journals Prognostic importance of flow cytometric immunophenotyping of 540 consecutive patients with B-cell chronic lymphocytic leukemia

Blood ◽  
1991 ◽  
Vol 78 (7) ◽  
pp. 1795-1802 ◽  
Author(s):  
CH Geisler ◽  
JK Larsen ◽  
NE Hansen ◽  
MM Hansen ◽  
BE Christensen ◽  
...  
Keyword(s):  
Chronic Lymphocytic Leukemia ◽  
B Cell ◽  
Fluorescence Intensity ◽  
Mononuclear Cells ◽  
Clinical Stage ◽  
Univariate Analysis ◽  
Lymphocytic Leukemia ◽  
Significant Difference ◽  
Prognostic Importance ◽  
Cell Chronic Lymphocytic Leukemia

Blood mononuclear cells from 540 newly diagnosed, unselected patients with B-cell chronic lymphocytic leukemia (CLL) were examined by immunofluorescence flow cytometry for a panel of surface membrane markers, including IgM and IgD, the monoclonal antibodies anti-CD3, -5, -20, -21, -22, -FMC7, and, for the final 125 patients, anti-CD23. There were 503 CD5+ and 37 CD5- cases. In the CD5+ cases, the cells typically expressed IgM, IgD, CD20, CD21, CD22, and CD23. In univariate analysis, age, clinical stage, IgM-fluorescence intensity, CD23, and FMC7 had significant prognostic importance, with high IgM-fluorescence intensity, high FMC7, and low CD23 expression being associated with a short survival. There was no significant difference in survival between 351 cases expressing IgMD and 55 cases expressing IgM without IgD, or between kappa and lambda light chain monoclonal cases. CD20, CD21, and CD22 had no prognostic importance. In Cox multiple regression analyses, age, CD23, IgM-fluorescence intensity, and clinical stage (International Workshop System) had independent prognostic importance. Thus, besides clinical variables, CD23 and IgM intensity might be useful prognostic markers in the management of CD5+, B-cell CLL. The survival of CD5- patients was on the borderline of being significantly shorter than that of CD5+ patients. The majority of the CD5- cases were FMC7+, CD23-, had strong IgM fluorescence, and had splenomegaly.

Sign in / Sign up

Export Citation Format

Share Document