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Supplementary Table 2 from CD38/CD31, the CCL3 and CCL4 Chemokines, and CD49d/Vascular Cell Adhesion Molecule-1 Are Interchained by Sequential Events Sustaining Chronic Lymphocytic Leukemia Cell Survival

Supplementary Figures 2-5 from CD38/CD31, the CCL3 and CCL4 Chemokines, and CD49d/Vascular Cell Adhesion Molecule-1 Are Interchained by Sequential Events Sustaining Chronic Lymphocytic Leukemia Cell Survival

Supplementary Table 1 from CD38/CD31, the CCL3 and CCL4 Chemokines, and CD49d/Vascular Cell Adhesion Molecule-1 Are Interchained by Sequential Events Sustaining Chronic Lymphocytic Leukemia Cell Survival

Supplementary Figure 1 from CD38/CD31, the CCL3 and CCL4 Chemokines, and CD49d/Vascular Cell Adhesion Molecule-1 Are Interchained by Sequential Events Sustaining Chronic Lymphocytic Leukemia Cell Survival

CD38 and CD49d are associated negative prognosticators in chronic lymphocytic leukemia (CLL). Despite evidence that both molecules are involved in interactions occurring between CLL and normal cells in the context of CLL-involved tissues, a functional link is still missing. Using gene expression profiles comparing CD38+CD49d+ versus CD38−CD49d− CLL cells, we showed overexpression of the CCL3 and CCL4 chemokines in cells from the former group. These chemokines were also up-regulated by CD38 signals in CLL; moreover, CCL3 was expressed by CLL cells from bone marrow biopsies (BMB) of CD38+CD49d+ but not CD38−CD49d− cases. High levels of CCR1 and, to a lesser extent, CCR5, the receptors for CCL3 and CCL4, were found in CLL-derived monocyte-macrophages. Consistently, CCL3 increased monocyte migration, and CD68+ macrophage infiltration was particularly high in BMB from CD38+CD49d+ CLL. Conditioned media from CCL3-stimulated macrophages induced endothelial cells to express vascular cell adhesion molecule-1 (VCAM-1), the CD49d ligand, likely through tumor necrosis factor α overproduction. These effects were apparent in BMB from CD38+CD49d+ CLL, where lymphoid infiltrates were characterized by a prominent meshwork of VCAM-1+ stromal/endothelial cells. Lastly, CD49d engagement by VCAM-1 transfectants increased viability of CD38+CD49d+ CLL cells. Altogether, CD38 and CD49d can be thought of as parts of a consecutive chain of events ultimately leading to improved survival of CLL cells. [Cancer Res 2009;69(9):4001–9]

Supplementary Tables 3-4 from CD38/CD31, the CCL3 and CCL4 Chemokines, and CD49d/Vascular Cell Adhesion Molecule-1 Are Interchained by Sequential Events Sustaining Chronic Lymphocytic Leukemia Cell Survival

Supplementary Methods and Materials, Figure Legends 1-5 from CD38/CD31, the CCL3 and CCL4 Chemokines, and CD49d/Vascular Cell Adhesion Molecule-1 Are Interchained by Sequential Events Sustaining Chronic Lymphocytic Leukemia Cell Survival

The effects of proliferative, apoptotic and anti-proliferative stimuli on interferon regulatory factor 4 (IRF4) expression by Reed-Sternberg (RS) cells were analysed using a panel of Hodgkin lymphoma (HL)-derived cell lines. IRF4 expressed by HL cells was consistently upregulated after CD40 engagement; IRF4 was downregulated by agonistic anti-CD95 antibodies in the FAS-sensitive HDLM-2 cells and after treatment with Adriamycin and Dacarbazine, two chemotherapic agents commonly used for HL treatment. These results demonstrated, for the first time, that IRF4 was up-modulated by CD40 engagement, and down-modulated by apoptotic and anti-proliferative signals, suggesting an involvement of IRF4 also in HL pathobiology.

We describe a case of human immunodeficiency virus-associated T-lymphoblastic leukemia/lymphoblastic lymphoma in a 43-year-old Italian man with a history of human immunodeficiency virus infection lasting 9 years. Immunoperoxidase stains showed that neoplastic cells were positive for CD3, TdT, CD45, CD10, CD1a, CD2, CD7, CD5, and CD43 (focal). The proliferation rate was approximately 70%, assessed by Ki-67/MIB-1 staining. Flow cytometry of the marrow aspirate revealed an intermediate/cortical T-lymphoblastic phenotype: negative for surface CD3 and positive for cytoplasmic CD3, CD1a, TdT, CD2, CD7, CD5, and CD8, with partial coexpression of dimCD4. Analysis of T-cell receptor gamma polymerase chain reaction products showed clonality. T-lymphoblastic leukemia/lymphoblastic lymphoma is a very rare occurrence in the clinical setting of human immunodeficiency virus infection. It is not listed in the World Health Organization classification of lymphomas associated with human immunodeficiency virus infection. Only 4 cases of human immunodeficiency virus-associated T-lymphoblastic leukemia/lymphoblastic lymphoma are reported in the current medical literature. (C) 2009 Elsevier Inc. All rights reserved.

Background and Objectives Ex vivo peripheral blood progenitor cell (PBPC) expansion has been proposed as a strategy to increase the number of haematopoietic progenitors available for cell transplantation. We have expanded CD34+ cells from PBPCs obtained from four patients with haematological malignancies and one patient with an Ewing's sarcoma. Materials and Methods Cells were expanded in the Dideco ‘Pluricell system’. After 12 days in culture, we evaluated cell phenotype, total nucleated cells, CD34+ fold increase, cell apoptosis and colony assay of expanded cells. Cell engraftment has been evaluated by transplanting two groups of irradiated non-obese diabetic/severe combined immunodeficient (NOD-SCID) mice with expanded and non-expanded cell populations. Results Total nucleated cells and CD34+ cells increased 59·5 and 4·0 times, respectively. The expanded cells were mainly constituted of myeloid and megakaryocytic cells. A significant increase in the number of colony-forming unit–granulocyte macrophage (CFU-GM) was observed in the CFU assay. Ten mice transplanted with expanded cells showed a best overall survival (80%) compared to 10 mice transplanted with non-expanded cells (20%). Human CD45+ cells were detected by flow cytometry and polymerase chain reaction in bone marrow and spleen of transplanted animals. The relative low engraftment level obtained with the expanded cells suggests a loss of SCID repopulating cells maybe due to cell differentiation during expansion. Conclusions We have demonstrated the feasibility of the ex vivo expansion of mobilized PBPCs from cancer patients, evidencing a clonal expansion of CFUs and the ability of the expanded cells to engraft the bone marrow and spleen of immunosuppressed mice. The differentiation of the CD34+ stem cell compartment could be further minimized by ameliorating the expansion conditions.

Classical Hodgkin's disease (HD) is a peculiar form of lymphoma characterized by a low frequency of tumor cells, the so-called Hodgkin (H) and Reed/Sternberg (RS) cells, embedded in a background of non-neoplastic (reactive) cells believed to be recruited and activated by H-RS cell-derived cytokines/chemokines. How these tumor cells can survive in such a seemingly hostile environment has confused researchers. We have previously identified interleukin (IL)-3 receptor (R) expression as a common feature of classical HD and unveiled the potential role of IL-3 as a growth and anti-apoptotic factor for H-RS cells. More then 90% of malignant cells of classical HD usually express the alpha chain of the IL-3R (IL-3R(alpha)), as evidenced by immunostaining of frozen sections and cell suspensions from neoplastic lymph nodes. Consistently, HD-derived cell lines (L428, KMH2, HDLM2 and L1236) express the alpha and beta chains that form IL-3R, both at the mRNA and protein level, with a molecular size of IL-3R(alpha) identical (70 kDa) to that expressed by human myeloid cells. Exogenous IL-3 promotes the growth of cultured H-RS cells, such an effect being potentiated by IL-9 and stem cell factor (SCF) co-stimulation, and is able to partially rescue tumor cells from apoptosis induced by serum deprivation. Finally, cultured H-RS cells are able to increase the production of IL-3 by pre-activated T cells, suggesting an involvement of IL-3/IL-3R interactions in the cellular growth of HD through paracrine mechanisms. This review will outline the biological activity of IL-3 and summarize the evidence indicating IL-3 as a growth and anti-apoptotic factor for H-RS cells in classical HD.

Purpose and Experimental Design: dCF (2′-deoxycoformycin) is a potent inhibitor of ADA (adenosine deaminase), an enzyme regulating intra- and extracellular concentrations of purine metabolites. ADA exists as cytosolic and extracellular forms, the latter colocalized on the cell surface with CD26. Once the surface expression of CD26 and ADA in a panel of cell lines and primary samples of T-cell leukemia/lymphoma was defined, we correlated this expression with the antiproliferative and apoptotic effect of dCF.Results: Surface expression of CD26 inversely correlated with the capability of dCF to inhibit cell growth and induce apoptosis both in T-cell lines and primary samples of T-cell malignancies. This conclusion was sustained by a decreased sensitivity to dCF-mediated proapoptotic and/or antiproliferative in vitro effects of: (a) leukemia/lymphoma T-cell lines expressing surface CD26/ADA complex; (b) primary CD26+ T cell malignancies; and (c) normal T cells (CD26+) as compared with tumor T cells (CD26−) in unpurified samples from three cases of T-cell receptor γδ+ T-cell malignancies characterized by a mixture of normal and neoplastic cells. This latter point was confirmed in vivo, in a patient affected by CD26− T-cell receptor γδ+ hepatosplenic γδ+ T-cell lymphomas treated on a compassionate basis with dCF. The inverse correlation between CD26 expression and sensitivity to dCF was also demonstrated in a lymphoblastic lymphoma case in which CD26 was expressed on circulating blasts at relapse but not at diagnosis, as well as in two H9 T-cell clones expressing or not expressing CD26 mRNA and protein.Conclusions: This study corroborates the notion of CD26 as a marker of poor prognosis for T-cell malignancies and delineates a role for CD26 as a predictor of poor response to dCF.

As a further extension of our research work focusing on the development of gold(III)-dithiocarbamato derivatives as potential anticancer agents, the new complexes [AuIIIX2(dtc-Sar-O(t-Bu))] (X = Cl (1)/Br (2)) are here reported. The compounds were characterized by means of FT-IR, ESI mass, and mono- and multidimensional NMR spectroscopy. In order to get further insights into the possible behavior under physiological conditions, their affinity toward selected model biomolecules was spectroscopically investigated in detail. In this regard, they seem to react very slowly with isolated dAMP (but not dGMP), forming a species identified as [AuIII(dtc-Sar-O(t-Bu))(dAMP-N1,C6NH)]+. In presence of GSH they undergo sudden reduction to the gold(I) counterpart [AuIII2(dtc-Sar-O(t-Bu))2], whereas only secondary interactions seem to occur when reacted with BSA. According to in vitro cytotoxicity studies, both complexes turned out to be highly effective toward all the human tumor cell lines evaluated (HeLa, L540 and U937), reporting IC50 values lower than cisplatin. Apoptosis was proved a major cell death mechanism and, accordingly, DNA fragmentation was observed. Remarkably, cell cycle progression was negligibly affected, thus supporting the hypothesis of a different mechanism of action from clinically-established platinum-based drugs.

The effects of proliferative, apoptotic and anti-proliferative stimuli on interferon regulatory factor 4 (IRF4) expression by Reed-Sternberg (RS) cells were analysed using a panel of Hodgkin lymphoma (HL)-derived cell lines. IRF4 expressed by HL cells was consistently upregulated after CD40 engagement; IRF4 was downregulated by agonistic anti-CD95 antibodies in the FAS-sensitive HDLM-2 cells and after treatment with Adriamycin and Dacarbazine, two chemotherapic agents commonly used for HL treatment. These results demonstrated, for the first time, that IRF4 was up-modulated by CD40 engagement, and down-modulated by apoptotic and anti-proliferative signals, suggesting an involvement of IRF4 also in HL pathobiology.

Background and Objectives Autologous peripheral blood stem cell transplantation has recently become a standard therapeutic approach to virus-related or infected haematological malignancies. Collection, manipulation, storage and thawing of leukapheresis products in this subset of patients require strict monitoring to prevent infection risk for operators and risk of contamination for other stored bags. Materials and Methods This is a non-randomized retrospective observational study. In the 2000–2002 period, a single bag freezing procedure was used for autologous peripheral blood stem cell transplantation. Bags were stored in tanks containing liquid and gas phase nitrogen. In 2002, the processing procedure was revised, and a second additional safety bag and a new storage tank containing jacketed liquid nitrogen have been used. Results A total of 524 bags were thawed, of which 121 processed with the single bag method and 403 with the double bag method. Forty-nine and 109 patients were infused respectively. The observed rupture rate with the single bag in liquid and gas phase nitrogen was 17 and 2·5%, respectively, against a rupture rate as little as 0·24% with the new methodology. Viability revealed levels of 84·4% ± 6·1% and 96·9% ± 2·4% for the single and double-bag respectively. This statistically significant (P

CD38 and CD49d are associated negative prognosticators in chronic lymphocytic leukemia (CLL). Despite evidence that both molecules are involved in interactions occurring between CLL and normal cells in the context of CLL-involved tissues, a functional link is still missing. Using gene expression profiles comparing CD38+CD49d+ versus CD38−CD49d− CLL cells, we showed overexpression of the CCL3 and CCL4 chemokines in cells from the former group. These chemokines were also up-regulated by CD38 signals in CLL; moreover, CCL3 was expressed by CLL cells from bone marrow biopsies (BMB) of CD38+CD49d+ but not CD38−CD49d− cases. High levels of CCR1 and, to a lesser extent, CCR5, the receptors for CCL3 and CCL4, were found in CLL-derived monocyte-macrophages. Consistently, CCL3 increased monocyte migration, and CD68+ macrophage infiltration was particularly high in BMB from CD38+CD49d+ CLL. Conditioned media from CCL3-stimulated macrophages induced endothelial cells to express vascular cell adhesion molecule-1 (VCAM-1), the CD49d ligand, likely through tumor necrosis factor α overproduction. These effects were apparent in BMB from CD38+CD49d+ CLL, where lymphoid infiltrates were characterized by a prominent meshwork of VCAM-1+ stromal/endothelial cells. Lastly, CD49d engagement by VCAM-1 transfectants increased viability of CD38+CD49d+ CLL cells. Altogether, CD38 and CD49d can be thought of as parts of a consecutive chain of events ultimately leading to improved survival of CLL cells. [Cancer Res 2009;69(9):4001–9]

The expression of CCL5/Rantes by Hodgkin (H) and Reed-Sternberg (RS) cells has been recently documented. In the present study we demonstrated that the CCL5 receptor (CCR5) is constitutively expressed by Hodgkin Lymphoma (HL)-derived cell lines (i.e. L-428, KM-H2, L-1236 and L-540) as shown by immunohistochemistry, flow cytometry and western blotting and also detected by immunohistochemistry on primary H-RS cells from lymph node tissues. sCD40L never significantly affected CCR5 expression, whereas a short exposure to doxorubicin down regulated its expression. CCR5 receptors on HL cell lines were functionally active, since neutralizing anti-CCL5 monoclonal antibodies inhibited basal proliferation of HL-derived cell lines and recombinant CCR5 ligands (CCL3/Mip-1 alpha, CCL4/Mip1 beta and CCL5/Rantes) increased their clonogenic growth. CCL5 secretion by L-1236, L-428 and KM-H2 cells was stimulated by CD40 engagement and also by coculturing L-1236 cells on primary stromal fibroblasts from HL-involved lymph nodes (HLF). Coculture experiments indicated that a direct contact of H-RS cells induces HLF cells to produce CCL5. Supernatants from L-1236, L-428 and KM-H2 cells stimulated migration of purified CD4+ T-cells and eosinophils in vitro. The migratory response to HL-cell lines supernatants was only partially neutralized (CD4+ cells: 70%; esinophils: 36%) by anti-CCL5 antibodies, reinforcing the notion that multiple chemokines are involved in the recruitment of nonmalignant reactive cells in HL tissues. Taken together, our results indicate a possible involvement of the CCR5/CCR5-ligands signaling in the regulation of H-RS cells growth and in the formation/maintenance of the typical tissue microenvironment of HL.

[Au(MSDT)Cl2] (dichloro[methyl N-(dithiocarboxy-kS,kS')-N-methylglicinato]gold(III)) and [Au(MSDT)Br2] (dibromo[methyl N-(dithiocarboxy-kS,kS')-N-methylglicinato]gold(III)) gold(III) dithiocarbamate derivatives are two newly synthesized gold(III) derivatives of methylsarcosinedithiocarbamate, containing a sulfur chelating ligand that is able to bind the metal center strongly, so preventing interactions with sulfur-containing enzymes; in fact these reactions are believed to be responsible for the nephrotoxicity induced by the platinum(II)-based drugs. Their activity has been compared with the well-known platinum-based anticancer agent cisplatin on a panel of acute myelogenous leukemia cell lines representing different French-American-British subtypes and in the Philadelphia-positive cell line K562. Both compounds suppressed, in a dose-dependent manner, colony formation in methylcellulose with ID50 values of about 10-fold lower than that of the reference drug. After a short exposure (18 h), our compounds, but not cisplatin, were able to: downregulate the antiapoptotic molecule Bcl-2, upregulate the proapoptotic molecule Bax and induce apoptosis, as determined by a strong induction of APO2.7 and phosphatidylserine exposure. Finally, after a 72-h exposure, both gold(III) dithiocarbamate derivatives determined modest cell cycle modifications, but induced DNA fragmentation in all myeloid cell lines tested. Altogether, our results indicate that these new gold(III) dithiocarbamate derivatives might represent novel potentially active drugs for the management of myeloid leukemia, able to combine cytostatic and apoptotic activity with reduced nephrotoxicity.

This work reports on the synthesis, characterization, and in vitro cytotoxic activity of some new platinum(II), palladium(II), and gold(III) derivatives of methylsarcosinedithiocarbamate and its S-methyl ester, to study their behavior as potential antitumor agents. The biological activity of these compounds, as determined by growth inhibition and apoptosis induction, has been investigated in both human leukemic promyelocites HL60 and human squamous cervical adenocarcinoma HeLa cell lines, and their activity has been compared to the well-known platinum-based anticancer agent cisplatin. On the basis of these experimental results, [Pd(MSDT)X]n (MSDT = methylsarcosinedithiocarbamate; X = Cl, Br) complexes show a strong dose-dependent growth inhibition of both HL60 and HeLa cells, with IC(50) values slightly higher than those recorded for cisplatin; moreover, [Au(MSDT)X(2)] activity appears significantly higher or, at least, comparable to that of the reference drug. Exposure of both cell lines to [Pd(MSDT)X]n and [Au(MSDT)X(2)] complexes induces apoptosis, as determined by an Apo2.7 assay.

Classic Hodgkin's Disease (cHD) is a lymphoid neoplasia characterized by a few malignant Hodgkin and Reed-Sternberg (H-RS) cells embedded in an abundant background of non-tumor cells. In this context, fibrosis is a common morphologic feature of HD lesions, being found more frequently in cHD subtypes. The clinical and histopathologic features of cHD are thought to be largely due to the effects of a wide variety of cytokines and chemokines primarily produced by H-RS cells, as well as by the surrounding reactive component. In the present review, first we propose three mechanisms putatively explaining fibroblast activation and fibrosis in HD: (1) unbalanced production of the pro-fibrogenic Th2 over Th1 cytokines; (2) production of TGF-beta, b-FGF and IL-13 by H-RS cells; (3) activation of fibroblasts by CD40L-expressing cells of the HD microenvironment. Second, we suggest some molecular pathways involving cytokines produced by HD-derived fibroblasts (SCF, IL-7, IL-6) supposedly responsible for H-RS proliferation and rescue from apoptosis. Finally, we describe the role of specific molecules produced by H-RS cells in the regulation of HD-derived fibroblast production of chemokines, in turn involved in T-lymphocytes and recruitment of eosinophils.

[Pd(MSDT)Cl] n palladium, chloro[methyl N -(dithiocarboxy-kS,kS')-N-methylglycinate], and [Pd(MSDT) Br] n palladium, bromo[methyl N -(dithiocarboxy-kS,kS')-N -methylglycinate], palladium (Pd)(II) derivatives are two newly synthesized Pd(II) derivatives of methylsarcosinedithiocarbamate (MSDT), containing a sulfur chelating ligand that is able to strongly bind the metal center, so preventing interactions with sulfur-containing enzymes. In fact, these reactions are believed to be responsible for the nephrotoxicity induced by platinum (II)-based drugs. Their activity has been evaluated in a panel of acute myeloid leukemia (AML) cell lines representing different French-American-British (FAB) subtypes and in the Philadelphia (Ph)-positive cell line K-562 and compared to cisplatin. Both compounds suppressed, in a dose-dependent manner, colony formation in methylcellulose with ID 50 values comparable to those of the reference drug cisplatin, excluding the ML-3 cell line (ID 50 10-fold lower than cisplatin). Exposure of HL-60, ML-3, NB-4, and THP-1 cell lines to a cytotoxic concentration of [Pd(MSDT)Br] n (5 μM) determined: downregulation of the antiapoptotic molecule Bcl-2, upregulation of the proapoptotic molecule Bax; apoptosis induction, as evaluated by AP02.7 and annexin V staining; mitochondrial membrane permeabilization; and DNA fragmentation. In ML-3 cells the Pd(II) complexes were more active than cisplatin in apoptosis induction. Finally, [Pd(MSDT)Br] n showed an inhibitory effect on clonogenic growth of hematopoietic progenitors (CFU-GM, CFU-GEMM, and BFU-E) with both ID 50 and ID 90 comparable to those of cisplatin. Remarkably, the Pd(II) complex was more potent in inhibiting the clonogenic growth of the less differentiated AML cell lines KG-1a, HL-60, NB-4, ML-3, and THP-1 (ID 50 ranging from 0.02 ± 0.001 to 0.52 ± 0.04 μM), compared to normal hematopoietic progenitors (ID 50 of 2.1 ± 0.1, 3.8 ± 0.4, and 2.5 ± 0.2 μM) for CFU-GEMM, BFU-E, and CFU-GM, respectively). These data suggest that leukemic cells of myelomonoblast lineage might represent a preferential target for its cytotoxic activity compared to normal committed hemopoietic progenitor cells. Altogether, our results indicate that these new Pd(II) dithiocarbamate derivatives might represent novel potentially active drugs for the management of some selected myeloid leukemia strains, able to conjugate cytostatic and apoptotic activity with reduced toxicity.