bluuebunny/crossref_metadata_2025_split huggingface dataset:
Induction of Growth Inhibition and Apoptosis by TZD18, a Novel Dual Ligand for PPAR alpha/gamma, in Human Ph-Positive ALL Cells In Vitro.
Authors: Elstner Elena , Liu Hongyu , Zang Chuanbing , Liu Dachuan , Xu Shunnan , Fenner Martin H. , Possinger Kurt 
Published: October 2019

Abstract
Peroxisome proliferator-activated receptors (PPARs) are ligand activated nuclear hormone receptors which play key roles in the differentiation and lipid metabolism of adipocytes. Recent data frequently indicated that PPAR ligands are also implicated in the growth inhibition, differentiation and apoptosis induction of several human cancers with diverse tissues. We previously showed that Pioglitazone (PGZ), a specific PPARgamma ligand and a member of the approved thiazolidinedione (TZD) class of anti-diabetic drugs, inhibited growth and induced apoptosis of human ALL cell lines including Ph-positive ALL cells (Zang et. al., Leukemia Research, 28:387, 2004). In this study, effects of a novel dual ligand specific for PPARalpha/gamma, TZD18 (MERCK, NJ, USA), on Ph-positive ALL cell lines, BV173, SD1 and Sup-B15 were examined. We noted that treatment of these cells with TZD18 resulted in growth inhibition in a dose-dependent manner which was associated with a G1 to S cell cycle arrest. This growth inhibition was much stronger than that of PGZ. However, this effect seemed not to be meditated through activation of PPARalpha or PPARgamma, since antagonists of PPARalpha or gamma could not reverse it. By studying the key regulators of cell cycle progression, we found that the expression of the cyclin dependent kinase inhibitor (CDKI) p27kip1, but not that of p21cip1, was enhanced whereas the expression of c-myc, cyclin D2, and cyclin dependent kinase 2 and 4 (CDK2 and CDK4) was decreased when these cells were treated with TZD18. Therefore, upregulation of p27kip1 and downregulation of cyclin Ds and CDKs may account for the G1 cell cycle arrest. Furthermore, a remarkable apoptosis induction was found in Ph-positive ALL cells treated with this dual ligand as measured by cell-death ELISA. No obvious alteration of bcl-2 levels but an upregulation of bax were observed in apoptotic cells. An activation of caspase-8 and caspase-9 by this ligand was also noticed. Of clinical importance, TZD18 enhanced the cytotoxic effect of Imatinib, a specific therapeutic agent for Ph-positive ALL. Overall, our findings strongly suggest that TZD18 may offer a new therapeutic agent for treatment of Ph-positive ALL in an adjuvant setting. (This study was supported by grants from Deutsche Jose Carreras Leukaemie-Stiftung and Deutsche Forschungsgemeinschaft to EE)
Leptomycin B Overcomes Imatinib Resistance Mediated by Stromal Cells and Mutant BCR-ABL in Philadelphia Chromosome-Positive Acute Lymphoblastic Leukemia Cells.
Authors: Tojo Arinobu , Izawa Kiyoko , Sekine Rieko , Nagamura Tokiko , Yoshida Minoru , Tsuji Takashi 
Published: October 2019

Abstract
Philadelphia chromosome-positive acute lymphoblastic leukemia (Ph-ALL) is one of the most intractable hematological malignancies, and readily acquires resistance to chemotherapeutic drugs including imatinib mesylate. We hypothesized that the adhesive interaction of Ph-ALL cells with bone marrow stromal cells might cause their escape from drug-induced apoptosis and subsequent minimal residual disease, resulting in the generation of a chemoresistant clone such as a clone harboring mutant BCR-ABL. To gain insight into this possibility and a novel strategy against imatinib resistance, we used two Ph-ALL cell lines designated as IMS-PhL1 (L1) and IMS-PhL2 (L2). L1 cells had wild type BCR-ABL, whereas L2 cells had Y253H mutant and revealed 10-fold or more resistance to imatinib, compared with L1 cells. The growth of L1 cells was autonomous and their spontaneous apoptosis was suppressed by co-culture with a murine bone marrow stromal cell line, HESS-5. In contrast, the sustained growth and survival of L2 cells was absolutely dependent on direct contact with HESS-5. Both cell lines adhered to and migrated beneath the HESS-5 cell layer, resulting in the formation of cobblestone areas (CA). While floating L1 cells were eradicated by 1 mM imatinib, a portion of adherent L1 cells could survive even at 10 μM imatinib. Similarly, L2 cells forming CA beneath the HESS-5 cell layer considerably resisted prolonged exposure to 10 μM imatinib. Leptomycin B (LMB), a potent inhibitor of CRM1/exportin-1, can trap BCR-ABL in the nucleus and can aggressively eliminate BCR-ABL+ cells in combination with imatinib (Wang et al., 2001). We tested LMB for its ability to eliminate CA or adherent Ph-ALL cells in combination with imatinb. The result for L2 cells was shown in Figure. Dramatically, combined use of 10 μM imatinib and 1 nM LMB for 7 days exerted a synergistic effect on reduction in the number of CA. L1 cells were also susceptible to the combination of imatinib and LMB. Our results suggest that nuclear entrapment of BCR-ABL may be a promising strategy for overcoming imatinib resistance mediated by stromal cells as well as a certain BCR-ABL mutant.
Figure Figure
Synergism between 17-AAG and Imatinib in Imatinib-Resistant CML Cells: Inhibition of P-Glycoprotein by 17-AAG as a New Mechanism of Increasing Imatinib Activity.
Authors: Radujkovic Aleksandar , Schad Moritz , Topaly Julian , Laufs Stephanie , Jauch Anna , Schultheis Beate , Melo Junia V. , Ho Anthony D. , Fruehauf Stefan , Zeller W. Jens 
Published: October 2019

Abstract
Chronic myelogenous leukemia (CML) has gained outstanding importance for targeted cancer therapy. Inhibition of the BCR-ABL tyrosine kinase by imatinib (STI571, Glivec, Gleevec) leads to reduced proliferation of CML cells in vitro and sustained hematological and cytogenetic responses in vivo. However, resistance has been observed after variable periods of imatinib monotherapy especially in advanced stages of disease and overexpression of the BCR-ABL protein is one of the mechanisms of imatinib resistance. As combination therapy may allow to overcome drug resistance, we were interested in the effect of combination treatment with imatinib and 17-allylamino-17-demethoxygeldanamycin (17-AAG), an inhibitor of the heat shock protein 90 (Hsp90) chaperone complex. Furthermore, a new mechanism of action of the heat Hsp90 inhibitor 17-AAG is brought to light giving implications for an additional benefit of a combination treatment of imatinb-resistant chronic myelogenous leukemia. In imatinib-sensitive CML cell lines, combination index values (CI) obtained using the method of Chou and Talalay indicated additive (CI = 1) or slightly antagonistic (CI > 1) effects following simultaneous treatment with imatinib and 17-AAG. In contrast, the agents acted synergistically in imatinib-resistant BCR-ABL overexpressing LAMA84-R cells (CI = 0.6 at 75 % growth inhibition level). Growth inhibition of CFU-GM colonies of primary CML cells obtained from 3 patients is stronger after combination treatment than after monotherapy and annexin V / propidium iodide staining showed a strong increase of the apoptotic cell fraction in CML cells treated for 48 hrs by the combination treatment as compared to treatment with each drug alone. In imatinib-resistant cells BCR-ABL mRNA levels and protein expression were increased compared to the imatinib-sensitive parental cell line which is consistent to our cytogenetic and multicolor FISH analyses revealing multiple genomic BCR-ABL copies in imatinib-resistant cells. Furthermore, in imatinib-resistant cells P-glycoprotein activity was increased. Single treatment with 17-AAG lowered BCR-ABL and increased Hsp70 protein levels in both cell lines as expected whereas combination treatment was even more effective in this respect. Interestingly, single treatment with 17-AAG also decreased P-glycoprotein activity in a dose-dependent fashion as confirmed by a rhodamine-123 exclusion assay. The synergistic effect of both drugs in imatinib-resistant cells may thus be explained by increased intracellular levels of imatinib following 17-AAG treatment. The relevance of this additional mechanism warrants further exploration in clinical studies.
Zoledronate Inhibits Leukemia Growth in Bone Marrow and Synergizes with Imatinib Mesylate Against Ph+ Primary Leukemic Cells.
Authors: Segawa Hidekazu , Kimura Shinya , Kuroda Junya , Yuasa Takeshi , Maekawa Taira 
Published: October 2019

Abstract
Imatinib mesylate has drastically changed Philadelphia chromosome positive (Ph+) leukemia treatment. However, remissions induced in advanced phase Ph+ leukemias tend to be short-lived even treated with imatinib. Combined therapy of imatinib with an agent which inhibits downstream signaling of BCR/ABL such as Ras is intriguing. Phase I studies of farnesyl transferase inhibitors (FTIs), which targeted farnesylation of Ras, with imatinib against Ph+ leukemias demonstrated limited combination effects. We previously reported that the third-generation bisphosphonates (BPs) zoledronate (ZOL) synergized with imatinib mesylate to inhibit Ph+ leukemia. BPs which inhibit both farnesylation and geranylgeranylation of Ras proteins may be more potent than FTIs. However, three important questions have remained unanswered to promote this attractive combination to a clinical trial. Concentrations of 20-30 μM ZOL are required in vitro to induce apoptosis in leukemic cell lines. However, peak serum concentrations after infusion of 4 mg ZOL were 1–2 μM. Therefore, it is important to investigate how ZOL achieves effective concentrations in vivo. The second is whether ZOL augments the effects of imatinib against not only cell lines but also primary cells from Ph+ leukemia patients. In addition, the safety of this combination therapy must be established, because the dose of ZOL used in our previous study was high compared with clinical doses administered for inhibition of bone lesions. To answer these questions, we herein investigated about the combination therapy of ZOL plus imatinib using NOD/SCID mice engrafted with primary Ph+ leukemic cells with or without mutations in the ATP binding site. ZOL inhibited the geranylgeranylation of Ras proteins of leukemic cells in bone marrow but not in peripheral blood, indicating that sufficient concentration of ZOL could be achieved in bone marrow. ZOL synergized with imatinib to enhance survival of mice engrafted with primary Ph+ leukemic cells without mutations and this combination was well-tolerated without any severe adverse effects. These findings suggest that ZOL inhibits leukemia growth in bone marrow and the combination of ZOL plus imatinib is a potential therapy for Ph+ leukemia patients.
Investigating the Biological and Molecular Mechanisms Underpinning the Engraftment/Selection Advantage Conferred to Hematopoietic Stem Cells by HOXB4.
Authors: Milsom Michael D. , Hollins Laura , Gagen Dorothy , Woolford Lorna B. , Fairbairn Leslie J. 
Published: October 2019

Abstract
We have recently demonstrated that co-expression of HOXB4 enables the enhanced delivery of HSC harbouring a second therapeutic trans-gene. Nonetheless, it is of great importance to elaborate the current knowledge about the mechanism of HOXB4 action in order to both evaluate the safety implications of its use in a clinical strategy, and to gain greater insight into the regulation of HSC self-renewal/expansion. To these ends we have performed an extensive in vitro analysis of the consequences of HOXB4 overexpression in primary murine BMC and in a murine multipotent myeloid progenitor cell line (FDCP-mix). We demonstrate for the first time in murine cells, that ectopic HOXB4 reduces the responsiveness of murine hematopoietic cells to differentiation stimuli. Furthermore, by performing a detailed investigation into the kinetics of FDCP-mix differentiation, we reveal that HOXB4 overexpression results in a specific differentiation delay as opposed to an outright block. We propose that an analogous delay is in operation in repopulating cells in order that the shift to increased assymetrical self-renewal, a requirement for stem cell expansion, is achieved. Notwithstanding this, it is clear that any perturbation in differentiation constitutes an increased risk of cellular transformation if this technology were transferred to a clinical setting. In order to further define the repercussions of ectopic HOXB4 delivery, we have developed a retroviral vector which encodes an activatable version of HOXB4. We have shown that this vector is able to mediate an in vitro differentiation delay in primary murine BMC and FDCP-mix as well as enable enhanced engraftment of BMC in vivo, both dependent upon the addition of the estrogen analogue; tamoxifen. Using this system, we are currently examining the effect of ectopic HOXB4 on the transcriptome of FDCP-mix cells, in addition to performing an in depth study into the biological mechanisms affected by HOXB4 overexpression in BMC in vivo. We envisage that these model systems will be particularly amenable to the manipulation required for target gene identification/validation.
Purification of Allogeneic Idiotype-Specific T Lymphocytes According to IFN-γ Production for Adoptive Immunotherapy in Multiple Myeloma Patients.
Authors: Curti Antonio , Pandolfi Simona , Isidori Alessandro , Baccarani Michele , Lemoli Roberto M. 
Published: October 2019

Abstract
In multiple myeloma (MM) adoptive immunotherapy through allogeneic donor lymphocyte infusions results in a well-documented graft versus myeloma effect, but it is associated with high incidence of graft versus host disease. The idiotype (Id) expressed by MM cells can be regarded as a tumor-specific antigen and it has been used for immunotherapy. To enhance antitumor immunity and reduce alloreactivity, allogeneic T cells were activated against tumor-derived Id and then purified according to IFN-γ production. Total peripheral blood mononuclear cells from healthy donors were incubated with autologous monocyte-derived dendritic cells and pulsed with patient-derived Id protein. Cells were maintained in serum-free medium and supplemented during the priming phase with IL-7 and IL-12. Subsequently, the T-cell culture was restimulated every 7 days with pulsed DCs in the presence of low doses of IL-2. After 2 or 3 stimulations, the percentage of IFN-γ-producing T cells was as high as 5–10%, whereas that observed in presence of not stimulated T-cells was undetectable. Based on their IFN-γ production, T cells were isolated by using a commercial immunomagnetic IFN-γ capture assay. The purity of enriched IFN-γ-producing T cells ranged between 60 and 90% as evaluated by flow cytometry. The yield was 60% of pre-selection IFN-γ positive T cells and cell viability after selection was 80%. Functionally, IFN-γ purified T cells showed better Id-specific proliferative response when compared both to not-stimulated T cells and to stimulated but not purified T cells. IFN-γ purified T cells were shown to be memory CD45RO+ T cells expressing HLA-DR, CD25, CD95 at high level, whereas the negative fraction was mainly composed of naive, poorly activated T cells. T-cell receptor spectratyping analysis demonstrated that IFN-γ positive T cells were represented by a pauciclonal cell population as compared to policlonal T cells obtained before cell culture. Moreover, addition of not-stimulated T cells resulted in a significant reduction of allogeneic CD34+-derived colony-forming capacity. Conversely, the absolute number of allogeneic total colony-forming units-cells was not affected by IFN-γ purified T cells.These data demonstrate that Id-specific T cells may be generated from healthy donors and significantly enriched on the basis of their IFN-γ production. Id-specific IFN-γ - purified T cells have better anti-Id response and reduced alloreactivity than unselected T cells.
Allogeneic Stem Cell Transplantation with Reduced-Intensity, Fludarabine-Based Conditioning in Relapsed and Refractory Hodgkin’s Disease: Low Transplant-Related Mortality and Impact of Intensity of Conditioning Regimen on Survival.
Authors: Anderlini Paolo , Saliba Rima , Donato Michele , Giralt Sergio , Andersson Borje , Ueno Nato T. , Khouri Issa F. , de Lima Marcos , Ippoliti Cindy , Champlin Richard E. 
Published: October 2019

Abstract
Forty patients with relapsed or refractory Hodgkin’s disease (HD) underwent allogeneic stem cell transplantation (allo-SCT) following a fludarabine-based conditioning regimen from an HLA-identical sibling (n=20) or a matched unrelated donor (n=20). The median age was 31 years (range 18-58). The median number of chemotherapy regimens received prior to allo-SCT was five (range 2-9). Thirty (75%) and thirty (75%) patients had received prior radiotherapy or a prior autologous SCT, respectively. The median time to progression after autologous SCT was nine months (3–52). Disease status at SCT was refractory relapse (n=14) or sensitive relapse (n=26). The conditioning regimens employed were fludarabine (25 mg/m sq IV x 5 days)-cyclophosphamide (1 g/m sq IV x 3 days) ± antithymocyte globulin (30 mg/kg IV x 3 days) (FC±ATG) (n=14), a less intensive regimen, and fludarabine (25 mg/m sq IV x 5 days) -melphalan (70 mg/m sq IV x 2 days) (FM) (n=26), a more intensive one. The two groups had similar demographics and prognostic factors. Chimerism studies indicated 100% donor-derived engraftment in 26/26 (100%) FM patients and in 9/13 (69%) evaluable FC±ATG patients. Day 100 and cumulative (18-month) transplant-related mortality (TRM) were 5 % and 22%, respectively for the whole group. There was a nonsignificant trend towards a lower cumulative TRM in the FM group (18% vs. 30% at 18 months, p=0.2). The cumulative incidence of acute (grade II-IV) GVHD was 38%. The cumulative incidence of chronic GVHD at 18 months was 69%. There was a trend for a lower relapse rate after the occurrence of GVHD, however, this was not statistically significant (hazard ratio 0.8; p= 0.6). Progression rates were similar in the FM and FC patients (53% vs. 57% respectively at 18 months, p=0.4). However, disease progression occurred later in FM patients (range 2–34 months) than in FC patients (range 0.7–13 months). In addition, with comparable follow-up time after progression, the FM group experienced a lower death rate after progression. Twenty-four patients (60%) are alive (fourteen in complete remission) with a median follow-up of 13 months (4–78). Sixteen patients expired (TRM n=8, disease progression n=8). FM patients had significantly better overall survival (73% vs. 39% at 18 months; p=0.03), and a trend towards better progression-free survival (37% vs. 21% at 18 months; p=0.2). We conclude that allo-SCT with fludarabine-based, less intensive conditioning from matched related and unrelated donors are feasible in high-risk HD patients with a low TRM. The intensity of the preparative regimen affects survival.
Minor Histocompatibility Antigen-Specific CD8+ Memory T Cells in Hematopoietic Compartments Survive the Immediate Non-Ablative and Ablative TBI Milieu.
Authors: Shatry Alwi M. , Levy Robert B. 
Published: October 2019

Abstract
Minor histocompatibility (MiHA) antigens induce CD8+ T-cell responses that mediate resistance to bone marrow engraftment in an MHC-identical, MiHA-disparate marrow transplant model, in which recipients are sensitized to donor antigens (BALB.B à B6BALB.B) prior to BMT. The H60 H antigen has been shown to dominate the immune response in B6 mice primed with BALB.B antigens (B6BALB.B). We initially sought to determine the distribution and partially characterize the phenotype of H60-specific CD8+ cells in the blood, spleen and marrow compartments of B6 mice primed with 3 x 107 BALB.B lymphoid cells and ≥3 weeks later boosted with 2 x 107 cells. An H60 tetramer (LTFNYRNL/H2-Kb) conjugated to PE was used to detect H60-specific CD8+ cells in these compartments. Eight days following the second immunization, the mean frequency of circulating H60-specific cells was 12.2% ± SE 0.88 of CD8+ cells (range: 5.6 – 20.5%). The frequency of splenic H60-specific CD8+ cells was equivalent to that of circulating antigen-specific cells, thus peripheral blood levels of H60-specific CD8+ cells appear to be representative of those resident in the spleen. Interestingly, in the marrow compartment, the frequency of H-60+ cells amongst the CD8 T cell population was higher compared to peripheral blood and spleen levels, suggesting that H60-specific CD8+ cells in this compartment may comprise both migrant cells from the periphery and resident cells in the marrow elicited during priming. In both BM and spleen, >90% of CD8+ H60+ cells expressed the memory phenotype(CD44+, Ly6C+) and as expected, did not express early activation markers (CD25, CD69). To mediate resistance to progenitor cell engraftment, H60-specific effector CD8+ cell must first survive the immediate post-BMT milieu in the hemopoietic compartments. To examine this question, B6BALB.B mice irradiated at 3.0, 6.0 and 9.0 Gy were analyzed 24 hours later for the presence of H60-specific CD8+ cells in the spleen and marrow compartments. Although there was an expected irradiation dose-dependent decrease in absolute numbers of CD8+ H60+ cells in the two compartments, there was a dose-dependent increase in percent of CD8+ T cells expressing the H60 TCR in both compartments. This observation indicates enhanced survival of these antigen-specific CD8+ memory T cells post-conditioning. Preliminary results indicate that 24h post-BMT into 9.0 Gy TBI recipients, there was BrDU uptake in marrow and splenic CD8+H60+ T cells in B6BALB.B transplanted with 1 x 107 BALB.B BM-TCD. Approximately 80% of CD8+H60+ T cells in the marrow compartment of primed recipients of BALB.B cells exhibited proliferation by BrDU uptake. Thus, donor MiHA-disparate marrow grafts elicit antigen-driven proliferation early post-BMT by CD8+ memory T cells in both compartments consistent with the potential importance of these cells in mediating resistance against progenitor engraftment across these MiHA differences.