bluuebunny/crossref_metadata_2025 huggingface dataset:
Lenograstim Treatment Following Cytotoxic Therapy in Tumor Patients.
Authors: Wolf Hans-Heinrich , Krohn Ilka , Sippel Christoph , Jordan Karin , Voigt Wieland , Krueger William
Published: October 2019

Incidence of febrile neutropenia following cytotoxic treatment of tumor patients can be reduced by prophylactic G-CSF treatment. We report results of a prospective open non-interventional multicenter observational trial in 2233 patients on lenograstim, a recombinant glycoprotein (rHuG-CSF) expressed and glycosylated in Chinese hamster ovary cells. In total 10 846 courses of chemotherapy followed by G-CSF treatment have been documented for either hematologic or oncologic malignancies (breast and ovarian cancer 4207 courses, Hodgkin’s disease and Non Hodgkin’s lymphoma 4381 courses, lung cancer 780 courses, other malignancies 1478 courses, respectively). Lenogratim was administered either interventionally or to less amount for prophylaxis of granulocytopenia; the mean daily dosage was 263 micrograms subcutaneously. A significant number of patients were treated for stage III or IV disease. Almost 1/3 of the patients (34,2%) were elderly aged at least 65 years old. The patients had not been hospitalized for cytotoxic therapy. Lenograstim treatment was started on day 6 (median 6,8 + 4,0) of chemotherapy regimens for almost 5 days (median duration of treatment 5,2 + 3,1 days). Median leukocyte count was 1,9 × 109/L when lenograstim treatment was started, and 5,9 × 109/L at the end of lenograstim treatment, respectively. Especially supporting the CHOP regimen in lymphoma patients lenograstim was administered prophylactically: in 39,5% of lymphoma patients, but only in 24,1% of patients with gynecologic tumors lenograstim treatment was performed for at least 5 days. The rate of infection or fever reported was 9,8% (7,8% in breast cancer patients, 9,0% in lymphoma patients, respectively). 87,1% of chemotherapy courses (87,5% in lymphoma patients and 86,6% in breast cancer patients) could be performed according to the protocol initially scheduled without reduction of dosage or delay of treatment intervals. We conclude that lenograstim was feasible and efficacious for prophylaxis of neutropenic fever in chemotherapy patients. In 2/3 of the patients daily lenograstim dosage of 263 micrograms was suitable to stimulate granulocytopoiesis after conventional cytotoxic therapy within 5 days. No serious adverse events possibly related to lenograstim were reported.
GlycoPEGylated Filgrastim XM22 Demonstrates Dose-Dependent Activity in Comparison to Pegfilgrastim in a Dose-Escalating Study in Healthy Volunteers after Body-Weight Dependent Single Dose.
Authors: Lubenau Heinz , Pokorny Rolf , Lopez-Lazaro Luis , Bias Peter , Wallin Bruce , Hinderer Walter , Maly Anne-Katrin , Seiberling Michael
Published: October 2019

XM22 is a glycoPEGylated recombinant human G-CSF that interacts with the G-CSF receptor by binding and activation. In pilot studies in non-neutropenic animals, XM22 demonstrated a similar PK/PD profile compared to pegfilgrastim. Because of its longer duration of action compared to filgrastim, XM22 is intended to be dosed once per cycle for the reduction of duration of severe neutropenia and incidence of febrile neutropenia in cancer patients undergoing chemotherapy.

METHODS: A single center, randomized, single-blind study of XM22, administered as single escalating body-weight (bw) dependent doses given by the s.c. route was conducted to assess safety, pharmacokinetic profile and pharmacodynamic properties based on absolute neutrophil count (ANC) and CD34+ cell count. Male or female healthy volunteers were assigned to one of 3 XM22 dose groups, at 25 μg/kg bw dose (n=8), at 50 μg/kg bw (n=15), and 100 μg/kg bw (n=15) compared to 100 μg/kg bw pegfilgrastim (n=15).

RESULTS: 53 healthy volunteers were enrolled in the three dose groups and completed 21 day follow-up. Injections were generally well-tolerated with no discontinuations due to adverse events or serious adverse events. XM22 at a dose of 100 μg/kg bw exhibited a PK profile with increased bioavailability and extended half life compared to the same dose of pegfilgrastim. ANC area over baseline effect curve (AOBEC) and CD34+ AOBEC were higher after treatment with XM22 100 μg/kg bw (6818.55 hr*neut./nl and 7340.16 hr*cell count/μl geometric mean respectively) compared to the same dose of pegfilgrastim. Antibody data will be presented.

CONCLUSIONS: Single body-weight dependent doses up to 100 mg/kg of XM22 administered to healthy volunteers were generally well-tolerated with regard to the expected side effect profile and demonstrated dose-dependent increases in absolute neutrophil count and CD34+ cell count. These data supplement results from another study in healthy volunteers in which XM22 was administered as a fixed dose.
New Classification of Chronic Graft Versus Host Disease: Added Clarity from the Consensus Diagnoses.
Authors: Arora Mukta , Nagaraj Sriharsha , Witte Judy , DeFor Todd E. , MacMillan Margaret L. , Burns Linda J. , Weisdorf Daniel J.
Published: October 2019

Chronic graft versus host disease (CGVHD) is classically defined as occurring more than 100 days after hematopoietic cell transplant (HCT). But, with newer donor types and transplant strategies being used [eg. non myeloablative (NMA) and reduced intensity conditioning (RIC)] the presentation of the syndrome is changing. Clinical syndromes resembling acute GVHD (aGVHD) are often recognized beyond 100 days after HCT; possibly misdiagnosed as CGVHD. Additionally, the old grading system of limited versus extensive GVHD was designed to identify patients likely to benefit from systemic immune suppression and does not capture the severity of organ involvement. The Diagnosis and Staging Working Group of the NIH Consensus Development Project on Criteria for Clinical Trials in chronic graft-versus-host-disease proposed criteria for diagnosis and assessment of overall CGVHD severity. We retrospectively reviewed 197 patients who underwent sibling donor transplant between January 2002 to December 2005 to assess the applicability of the new criteria in predicting survival and transplant related mortality (TRM). Fifty four patients were diagnosed with CGVHD [cumulative incidence 28% (95% CI 22–34%)]. Of these, 8 [cumulative incidence 4% (95% CI 2–6%)] were reclassified as Late aGVHD; 31 [cumulative incidence 16% (95% CI 11–21%) had classic CGVHD and 15 [cumulative incidence 8% (95% CI 5–11%] had Overlap syndrome. Forty six patients with Overlap syndrome and classic CGVHD were graded. Six patients (13%) had Mild, 29 (63%) Moderate and 11 (24%) Severe CGVHD. After a median follow up of 35 months (range 4.3–59.2mo), 3 year probability of overall survival was significantly worse in Late aGVHD [25%(95% confidence interval 4–56%)] as compared to Overlap [87%(56–96%)] or CGVHD [75%(54–87%), p=0.001].). Amongst patients with Overlap and CGVHD (n = 46), similar survival was seen between mild or moderate disease. However, patients with severe disease had a trend to worse survival. [Mild-moderate 3 year probability of survival 85% (68–94%) versus Severe 57%(21–82%)]. Additonally, 3 year cumulative incidence of TRM was higher in patients with Late aGVHD [38% (9–67%)] versus Overlap syndrome [13%(0–27%)] or CGVHD [7%(0–13%)], p= 0.003. A trend towards greater TRM was seen in patients with severe GVHD [18%(0–36] versus mild+ moderate GVHD [6%(0–14%)]. This analysis indicates that the consensus guidelines are applicable to related donor HCT recipients and more reliably predicts survival and TRM than older classification of CGVHD. Prospective validation and testing in alternative donor cohorts is still needed before widespread acceptance is justified.
The Optimal Processing Time of Adipose-Derived Mesenchymal Stem Cell after Lipoaspiration.
Authors: Jang Jun Ho , Kim Kihyun , Jung Chul Won , Park Keon Woo
Published: October 2019

Background: Our aims were to characterize the phenotype of these cells, to investigate their immunoregulatory properties in vitro, and to determine the optimal processing time of AD-MSC after lipoaspiration.

Methods: ASCs were isolated from lipoaspirated adipose tissues by treatment of collagenase A and cultured in a Dulbecco’s Modified Eagle’s Medium (DMEM). To evaluate the optimal processing time of ASCs, the nucleated cells were processed by collagenase treatment at 3, 6, 9, 12, 15, 24, 30 and 36 hours after lipoaspiration respectively. To know the characteristics of ASCs, the expression of cell surface antigens was analyzed by flow cytometry, and the proliferation potentials were estimated by colony forming abilities or capacities of population doubling. The differentiation potentials into adipocytes or osteoblasts were confirmed by accumulation of neutral lipid vacuoles stained with Oil-red O and expression of alkaline phosphatases.

Results: When the nucleated cells were isolated by collagenase treatment after lipoaspiration, the mean cell yield was about 3.1 × 106 or 1.2 × 106cells per gram of lipoaspirate (n=8) processed. Nucleated cell counts were increased with time but plateau between 15h and 24h after initial processing and decreased after 24h. Flowcytometric analysis showed that Adipose tissue-derived stem cells (ASCs) have a marker expression that is similar to that of bone marrow stromal cells (BMSCs). ASCs expressed CD44, CD73, CD90, and CD105 and were absent for CD14, CD31 and CD45 expression. When primary cells were plated at 50 or 1000 cells/cm2 on 6-well plates, cumulative population doublings were about 50 times until passage 7 or 13 (approximately 130 days), respectively, and ASCs expanded to 1018 cells. ASCs were multipotent, differentiating along the adipocyte and osteoblast lineages. ASCs did not provoke in vitro alloreactivity of incompatable lymphocytes and, moreover, suppressed mixed lymphocyte reaction (MLR) and lymphocyte proliferative response to mitogen.

Conclusion: The optimal processing time for AD-MSCs from lipoaspirates should before 24h after removal RBC and AD-MSCs can be a alternate source of multilineage MSCs for clinical use such as tissue repair and transplantation.
GM-CSF Stimulates IL-8 Expression in Human Bone Marrow Stromal Cells Via Activation of NADPH Oxidases.
Authors: Wu Shuling , Enders Sven , Burmeister Thomas , Nowak Daniel , Reinwald Mark , Thiel Eckhard
Published: October 2019

The self-renewal, proliferation, and mobilization of hematopoietic progenitor cells (HPC) depend on complex interactions involving cytokines and bone marrow (BM) stromal microenvironment. Little is known about the effect of mobilizing agents such as granulocyte-macrophage colony-stimulating factor (GM-CSF) on BM stromal cells and their contribution to the mobilization of peripheral blood stem cells. In the present study we show that, 3 hours after addition, GM-CSF distinctly upregulated IL-8 mRNA expression and protein secretion in cultured human BM stromal cells. Furthermore, GM-CSF stimulated the generation of reactive oxygen species (ROS) in a dose- and time-dependent manner. Intracellular levels of ROS correlated with the extent of IL-8 production and IL-8 promotor activity as determined by luciferase reporter assay. Using western blot analysis, we showed that BM stromal cells expressed both phagocytic and non-phagocytic NAD(P)H oxidase components such as gp91phox, NOX1, p22phox, p47phox, p67phox, and NOXO1, suggesting that the upregulation of IL-8 expression induced by GM-CSF may be associated with the activation of stromal cell NADPH oxidase activity. GM-CSF directly stimulated the phosphorylation of p47phox, a key event in NAD(P)H oxidase activation. Inhibitors of the ERK1/2 and AKT pathways abrogated GM-CSF-induced phosphorylation of p47phox, indicating that GM-CSF-induced phosphorylation of p47phox is mediated by these pathways in BM stromal cells. Accordingly, the transactivation of IL-8 promotor activity induced by GM-CSF was suppressed by these inhibitors and the antioxidants diphenylene iodonium (DPI) and N-acetyl-l-cysteine (NAC). In conclusion, these findings show that GM-CSF elicits the activation of the redox-sensitive signaling via ROS, resulting in stromal cell function such as cytokine production and therefore possibly impacts on hematopoiesis.
Phase 1 Single Agent Antitumor Activity of Twice Weekly Consecutive Day Dosing of the Proteasome Inhibitor Carfilzomib (PR-171) in Hematologic Malignancies.
Authors: Alsina Melissa , Trudel Suzanne , Vallone Marcy , Molineaux Christopher , Kunkel Lori , Goy Andre
Published: October 2019

Carfilzomib (CFZ) is a structurally- and mechanistically-novel proteasome inhibitor of peptide epoxyketone class that exhibits a high level of selectivity for the unique N-terminal threonine active sites within the proteasome. CFZ is similar to bortezomib (BTZ,Velcade®), in that it is a potent inhibitor of the proteasome chymotrypsin-like activity, but unlike BTZ, CFZ has shown minimal cross-reactivity with the other catalytic sites within the proteasome or across other protease classes. Preclinical human tumor xenograft models indicated that consecutive day (D1, D2) dosing resulting in 48 hours of proteasome suppression was superior to split dosing (D1, D4). This phase 1 sequential dose escalation trial tested twice weekly consecutive day dosing (48 hour proteasome inhibition) with carfilzomib. Carfilzomib was administered as an IVP on a 28 day cycle (D1, 2, 8, 9, 15 and 16 with 12 days rest) at doses from 1.2 mg/m2 to 27 mg/m2. Patients with Multiple myeloma (MM), non-Hodgkin’s Lymphoma (NHL) that included mantle cell lymphoma (MCL), Hodgkin’s disease (HD), or Waldenström’s macroglobulinemia (WM) were eligible if they had relapsed after at least 2 prior therapies. A total of 37 subjects have been treated. The minimal effective dose (MED) was 15 mg/m2, and maximum (80%) proteasome inhibition was achieved in peripheral blood and mononuclear cells at this dose. Five objective responses have occurred in 16 patients (13 MM, 3 MCL) enrolled at ≥ the MED: 4 MM partial response (PR), 1 MM minimal responses (MR). The responses have been durable (134 to 392 days) and occurred in patients who had failed bortezomib, immunomodulatory agents and stem cell transplant. Six additional patients have had stable disease (SD): 2 MM, 1 T NHL, 2 MCL, 1 B NHL. Dose limiting toxicity reported at 27 mg/m2 included a hypoxic event and Grade 2 thrombocytopenia increasing to Grade 4. A reversible Grade 2 creatinine D2 was reported in 3/5 MM patients treated at 27 mg/m2 and was associated with a rapid decline in M-protein without evidence of tumor lysis syndrome and the event did not occur on rechallenge. Cyclic thrombocytopenia was rapidly reversible and painful peripheral neuropathy was not reported. This study demonstrates that consecutive CFZ dosing leading to sustained proteasome inhibition is well tolerated on a weekly basis. The selectivity of epoxyketone class of proteasome inhibitors may provide a wide therapeutic index. Patients have remained on therapy for over a year, and furthermore, antitumor responses in heavily pretreated MM patients suggest non-cross resistant activity. Phase 2 trials in relapse and refractory MM and relapse solid tumors are proceeding with this schedule.
Functional Alterations of Lin−CD34+CD38+ Progenitors in Chronic Myelomonocytic Leukaemia and on Progression to Acute Leukaemia.
Authors: Sun Qian , So Chi-Chiu , Yip Sze-Fai , Wan Thomas S.K. , Ma Edmond Shiu Kwan , Chan LiChong
Published: October 2019

Chronic myelomonocytic leukaemia (CMML) is a clonal bone marrow stem cell disorder based on the presence of trilineage involvement, the association of myelodysplastic and myeloproliferative features and its ability to transform into acute myeloid leukaemia. The objectives of our study are to identify the cell population and its functional characteristics involved in evolution from CMML phase to acute myeloid leukaemia. We analysed Lin−CD34+ stem/progenitor population and performed cell proliferation, apoptotic assays, self-renewal ability and differentiation potential studies in purified populations of Lin−CD34+CD38− stem cells and Lin−CD34+CD38+ committed progenitors from peripheral blood of 16 patients with CMML and in six of the 16 after transformation to acute myeloid leukaemia (AML-t). We observed an expansion of the stem cell/progenitor pool (Lin−CD34+ cells) in AML-t comprising mainly of Lin−CD34+CD38+ committed progenitors within Lin−CD34+ cells. The Lin−CD34+CD38+ committed progenitors displayed highly proliferative activity in CMML and in AML-t; and additionally acquired resistance to apotosis and myeloid colony self-renewing ability in AML-t. Impairment of dendritic cell (DC) differentiation was observed with complete block in AML-t. Our findings suggest Lin−CD34+CD38+ committed progenitors instead of Lin−CD34+CD38− stem cells could be the target(s) of secondary genetic lesions underpinning progression from CMML to AML. These results have implications for the further study of the biology of leukaemic transformation and the design of new strategies for the effective treatment of CMML.
The Establishment and Characterization of a Human Acute Myelocytic Leukemia Cell Line, SH-2 Carrying t(16;17))(q24;q12) Translocation.
Authors: Qiu Huiying , Xue Yongquan , Pan Jinlan , Wu Yafang , Wang Yong
Published: October 2019

We report the establishment of a novel cell line from a patient with acute myelocytic leukemia. The initial growth of this cell line was dependent on the presence of the stromal cell and cytokine interleukine 3. Subsequently, a stroma and cytokine-independent cell line was established, named SH-2. The cell line has proliferated continuously in vitro for more than 24 months. The SH-2 cell line showed identical chromosomal alterations to that of the initial bone marrow aspiration specimen, demonstrated a t(16;17))(q24;q12) translocation accompanied by deletions of y and 17 and triple 19.Chromosome painting FISH and Multi-FISH conformed the karyotype.SH-2 cells expressed myeloid antigens CD13, CD33 and MPO, SH-2 co-expressed NK markers CD16 and CD56. The loss of one p53 allele were proven by chromosome painting, FISH. A point mutation of CAG to CAT at codon 576 of extron 5 in another p53 allele was found by direct sequencing of DNA. Neither Epstein-Barr virus nor mycoplasma was not detected in SH-2 cells. DNA fingerprinting confirmed the authenticity of the cell line. Cytokines of GM-CSF,IL-3,IL-6,SCF,IL-4 and IL-5 can improve the proliferation of SH-2. SH-2 has colony formality and tumorigenic capacity in nude mice. The breakpoints of chromosomes 16 and 17 were localized RP11-356C4(16q24.3)and between BAC clone RP1-161P9 and RP11-47L3 corresponding to 17q12. The establishment of an myelocytic leukemia cell line with t(16;17)(q24;q12) could be valuable for the study of leukemogenesis and for the research of cloning the new gene involved in the t(16;17)(q24;q12) translocation.
Preferential Hypermethylation of the DKK-1 Promoter in Core-Binding Factor Leukaemia.
Authors: Suzuki Rikio , Onizuka Makoto , Kojima Minoru , Shimada Masako , Fukagawa Satomi , Tsuboi Kosuke , Kobayashi Hiroyuki , Shintani Ayumi , Ogawa Yoshiaki , Kawada Hiroshi , Hotta Tomomitsu , Ando Kiyoshi
Published: October 2019

The Dickkopf-1 (DKK-1) gene product is an extracellular Wnt inhibitor. Hypermethylation of the DKK-1 promoter results in transcriptional silencing and may play an important role in cancer development. Here, we investigated hypermethylation of the DKK-1 promoter in patients with acute myeloid leukaemia (AML), especially core-binding factor (CBF) leukaemia. The methylation status of DKK-1 was analyzed by methylation-specific polymerase chain reaction in 47 patients with AML. DKK-1 methylation was found in 14 (29.8%) of the patients, and more frequently in those with CBF leukaemia (6 of 12 patients), than in those with acute promyelocytic leukaemia (0 of 6 patients) (P = .03). In contrast, Wnt inhibitory factor-1 methylation was found in acute promyelocytic leukaemia (4 of 6 patients) but not in CBF leukaemia (0 of 12 patients) (P = .001). Multivariate analyses suggested that DKK-1 methylation was a risk factor for poorer overall survival. Sequential analysis using 4 paired samples obtained at diagnosis and relapse suggested that DKK-1 methylation was involved in the progression of leukaemia. DKK-1 methylation may therefore be involved in leukaemogenesis, especially in CBF leukaemia, and may be a useful prognostic marker in AML.
The Epigenetic Effect of Phenylhexyl Isothiocyanate on Histone Acetylation in Patients with Acute Leukemia and Non-Leukemic Disorders.
Authors: Xiao Liyun , Ma Xudong , Huang Yiqun , Zhen Ruiji , Ye Xiaoling , Chiao Jen-Wei , Liu Delong
Published: October 2019

Phenyhexyl isothiocyanate (PHI) has been shown to inhibit histone deacetylation in leukemia cell lines in vitro and in animal models. In this study we examined the histone acetylation status in leukemia cells from patients with acute leukemia as well as in non-leukemia cells. From January to September 2006, 19 patients with newly diagnosed acute leukemia were enrolled. 12 patients had AML, 7 had ALL. The age ranged between 2 months to 65 years old. There were 12 males and 7 females among them. 5 non- leukemia patients were also enrolled, with 1 anemia, 1 infection disease, 2 ITP, and 1 healthy volunteer. Mononuclear cells were isolated from patients’ specimen. Apoptosis of the cells and histone acetylation of H3 and H4 were characterized pre- and post- PHI treatment. The expression of acetylated H3 and H4 in acute leukemia cells was decreased, as compared to those in non-leukemia cells. Increased apoptosis was seen in the acute leukemia cells after PHI treatment of the primary culture of the leukemia cells for 3 and 7 hours. The effect of PHI on the leukemia cells from patients was time- and dose-dependent. There was a significant increase of PHI effect on leukemia cells versus non-leukemia cells (p<0.05), suggesting that PHI may have preferential activity against leukemia cells. This study presents the first evidence of PHI with preferential activity against leukemia cells over non-leukemia cells from human subjects. Clinical trial is warranted to study PHI in leukemia patients.
Decreased Expression of the Histone Methyltransferase SUV39H1 in AML Cells Reactivates Hypermethylated Tumor Suppressor p15INK4B in the Absence of Promoter Demethylation.
Authors: Lakshmikuttyamma Ashakumary , Scott Stuart , Geyer C. Ronald , DeCoteau John F.
Published: October 2019

Re-expression of hypermethylated tumor suppressor genes using epigenetic modifiers, such as DNA methyltransferase (DNMT) and histone deacetylase (HDAC) inhibitors, occurs by a mechanism whereby promoter demethylation is the dominant event. In support of this model, we found that the DNMT inhibitor 5-Aza-2-deoxycytidine (decitabine) induces expression of the tumor suppressor gene p15INK4B (p15) in AML cells with hypermethylated p15 promoters. Re-expression of p15 by decitabine is associated with decreases in p15 promoter methylation and histone H3 lysine 9 (H3K9) methylation and increases in H3K9 acetylation. DNA methylation is linked to H3K9 methylation through the DNA methyl binding protein MeCP2, which associates with DNMTs and H3K9 methyltransferases. Using chromatin immunoprecipitaton (ChIP) assays, we confirmed that MeCP2, DNMT1 and the H3K9 methylatransferase SUV39H1 interact with the methylated p15 promoter and that this interaction is reduced by decitabine. To determine whether promoter demethylation is also dominant to H3K9 demethylation, we monitored p15 re-expression in the presence of SUV39H1 shRNA alone and in combination with decitabine. SUV39H1 shRNA induces p15 expression and H3K9 demethylation, however it does not affect p15 promoter methylation. These results are in contrast to the HDAC inhibitor trichostatin A (TSA), which cannot induce p15 re-expression. SUV39H1 shRNA induced p15 expression and H3K9 demethylation are also enhanced by co-treatment with decitabine or TSA. Surprisingly, co-treatment with decitabine and SUV39H1 shRNA partially reverses decitabine induced promoter demethylation. Using ChIP assays we show that SUV39H1 shRNA increases the amount of the histone H3K9 dimethytransferase G9a and DNMT1 associated with the p15 promoter. Increased levels of G9a at the p15 promoter would enhance promoter methylation since G9a stimulates DNMT1 activity. Our results demonstrate that hypermethylated p15 can be reactivated in AML cells by an initial event that involves H3K9 demethylation. In addition, we found that the SUV39H1 inhibitor chaetocin induces p15 in AML cells with hypermethylated p15 promoters. Therefore, H3K9 methylatransferases represent novel therapeutic targets for developing inhibitors to reactivate the expression of hypermethylated genes.
The Significance of MicroRNA-155 in Lymphocytic Malignancy.
Authors: Banerjee Arnob , Reiner Steven
Published: October 2019

Oncogenes and tumor suppressor genes implicated in lymphocytic malignancy mainly encode proteins, but more recent studies have identified specific micro-RNA (miR) molecules that also have an important role in the transformation of lymphocytes. MiRs are non-coding RNA molecules of approximately 22 nucleotides that regulate gene expression by targeting specific mRNAs for silencing or degradation. Certain miR’s, termed oncomiRs, have been shown to play a role in malignant transformation. MiR-155 is one of the first described onco-miRs, with high levels of expression observed in biopsy samples from patients with Hodgkin’s lymphoma, primary mediastinal B cell lymphoma, and diffuse large B cell lymphoma. MiR-155 was demonstrated to have a causal role in B cell transformation in transgenic mice with forced overexpression of miR-155 in the B cell lineage. The transgenic mice develop fatal aggressive B cell malignancies in the first six months of life. The mechanism of miR-155 in lymphocytic transformation remains unclear. Some insight into the physiologic function of miR-155 was gained using mice with a deletion of the gene encoding miR-155. The mice have defects in germinal center formation and in cytokine production by B and T cell lineages. We have used combined gain and loss of function approaches to study the function of miR-155 in T helper cells. Our experiments have demonstrated effects on T helper cell differentiation that are dependent on both the level and the timing of miR-155 expression. The cellular phenotypes we have observed, in combination with computational target prediction have allowed us to identify several promising mRNA targets which are expressed in both T cells and B cells. Currently we are investigating whether any of the target mRNA candidates we have identified contribute to the oncogenic potential of miR-155. We have screened several lymphocyte cell lines for levels of miR-155 expression, and have identified lines in which miR-155 is highly expressed. We are using a loss of function approach to test the dependence of these lines on miR-155 expression. The relative contribution of each candidate target mRNA from our previous studies is being assessed by forced expression of the mRNA in the presence of miR-155 expression and by knockdown in the presence of miR-155 antagonism. Further functional studies and investigation of mRNA targets will help to elucidate both the physiologic role of miR-155 in lymphocytes and its role in lymphocytic malignancy.
Variation of Sorcin’s Expression in the Reversion of Multidrug Resistance of K562/A02 Leukemic Line with Tetrandrine.
Authors: Chen Bao-An Chen , Li Jing , Ding Jia-Hua , Gao Chong , Sun Yun-Yu , Gao Feng , Wang Jun , Zhao Gang , Chen Ning-Na , Chen Jian , Pei Xiao-Ping
Published: October 2019

Objective This paper aims to investigate the reversal multiple of daunomycin(DNR) with different concentration of tetrandrine(tet) at different time, to study the variation of Soluble resistance related calcium binding protein (sorcin)’s expression in the reversion of multidrug resistance of K562/A02 leukemic line with different concentration of tet at different time, and to provide new theoretic evidence for the clinical application of tet as resistence modifying agents.

Method

The reversal multiple of DNR with different concentration of tet at different time was assayed by MTT (concentration of tet is 0.5mg/l or1mg/l or 2mg/l, incubation time is 48h or 72h). The variation of the gene’s expression of sorcin with different concentration of tet at different time wasassayed by RT-PCR(grouping like MTT).

Result

MTT analysis demonstrated that the reversal concentration was 1.81(0.5mg/l,48h),3.62(1.0mg/l,48h),6.14(2.0mg/l,48h),2.8(0.5mg/l,72h),(1.0mg/l,48h),7.12(2.0mg/l,72h) respectively. RT-PCR analysis demonstrated that sorcin gene was lowly displayed in K562 cells and highly displayed in K562/A02 cells.

The variation was first accentuation and then attenuation with the concentration accrescence of tet, this tendency had no obviously difference between 48h and 72h.

Conclusion

1 The result of the experiment shows that tet may reverse multidrug resistance of K562/A02 cells by the down regulation of the expression of sorcin gene and proteinum;

2. It may have the concentration dependence and the time dependence in the reversion of multidrug resistance of K562/A02 cells with tet.
Lycorine Inhibits the Proliferation of HL-60 Cell Line through Up-Regulation of p21.
Authors: Liu Jing , Shi Bi-Wei , Hu Ji-Liang , Hu Wei-Xin
Published: October 2019

In previous studies we found that lycorine had broad antitumor effects and tumor cells were more sensitive to lycorine than non-tumor cells. We also showed that the mitochondrial and cell death receptor pathways were involved in lycorine-induced apoptosis of HL-60 cells. To further explore the molecular mechanism by which lycorine inhibits cell proliferation, the gene chip technique was used to detect the difference of gene expression patterns between 5.0 μM lycorine-treated HL-60 cells and control. The chip included 494 genes associated with apoptosis and proliferation. This data set revealed a significant change of 91 genes in expression, with 79 being up-regulated and 12 down-regulated. In particular the tumor suppressor gene p21 was up-regulated 9.3 folds in the lycorine-treated group. Consistent with this finding, RT-PCR and Western blotting confirmed the up-regulation of p21 gene and its protein in HL-60 cells treated with 5.0 μM lycorine for 24 h. The levels of p21 protein and its downstream cell cycle molecules Cdc2, Cdk2 and cyclin E were assessed after HL-60 cells were incubated with 0, 1.25, 2.5 and 5.0 μM lycorine. The results showed that the expression of Cdc2, Cdk2 and cyclin E was down-regulated, while that of p21 was up-regulated. Thus, lycorine could induce cell cycle arrest by interfering with cell cycle signaling pathways through up-regulation of p21 and concomitant down-regulation of Cdc2, Cdk2 and cyclin E in HL-60 cells.
P-Glycoprotein Is Downregulated in KG1a Primitive Leukaemia Cells by LDL Cholesterol Deprivation and by HMG-CoA Reductase Inhibitors.
Authors: Connelly-Smith Laura , Pattinson Joanne , Grundy Martin , Shang Shili , Seedhouse Claire , Russell Nigel , Pallis Monica
Published: October 2019

P-glycoprotein (pgp) is a membrane transporter encoded by the multidrug resistance (MDR1, ABCB1) gene. Pgp is a poor prognostic factor in elderly patients with acute myeloid leukaemia (AML). In addition to its role in drug efflux, pgp has been implicated in cellular cholesterol homeostasis. We investigated the effects of exogenous cholesterol removal on pgp expression and function. KG1a drug-naïve, primitive leukaemia cells were cultured in serum free medium with or without the addition of low density lipoprotein (LDL) cholesterol. After 72 hours pgp expression and function was assessed by flow cytometry and total cholesterol content of the KG1a cells was determined by the Amplex Red® cholesterol assay. The addition of clinically available cholesterol lowering agents, HMG-CoA (3-hydroxy-3-methylglutaryl coenzyme A) reductase inhibitors to KG1a cells was also assessed. There was a 39% (SEM 8.3% P=0.03) decrease in pgp protein expression after 3 days of serum free culture without HMG-CoA reductase inhibitors. Message was decreased by 40% (P=0.01) and pgp function was also reduced by 40% (P=0.005). The addition of low density lipoprotein (LDL) cholesterol restored pgp expression to 86% of the basal value. The addition of a HMG-CoA reductase inhibitor to KG1a cells in serum free culture resulted in a further 26% (lovastatin, P=0.03) and 16% (pravastatin, P=0.05) reduction in pgp respectively. Lovastatin also significantly reduced cellular cholesterol levels by 47% (P=0.002) under serum free conditions. Furthermore, the toxicity of the pgp substrate drug daunorubicin was significantly enhanced following lovastatin pre-culture (P=0.04). We conclude that LDL/cholesterol contributes to pgp expression and chemoresistance in primitive leukaemia cells. The use of HMG-CoA reductase inhibitors may be of clinical value in lowering pgp expression in AML.
Synergistic Anti-Leukemic Effect of Protein Kinase A Activator and PPARγ Agonist Is Independent of Bcl-2 Status.
Authors: Erikstein Bjarte S. , Tronstad Karl J. , McCormack Emmet , Schwede Frank , Bergen Rolf , Gjertsen Bjorn T.
Published: October 2019

The fatty acid derivate tetradecylthioacetic acid (TTA) and 8-CPT-cAMP have previously been shown to have anti-proliferative activities through activation of PPARγ and effects on the mitochondrial metabolism, respectively. Based on our previous work, we hypothesized that these two agents have a converging point of action. The combined effect of these two compounds was investigated in acute myelogenous cell lines (IPC-81 and HL-60), including IPC-81 clones over expressing either Bcl-2 or inducible cAMP early transcription repressors (ICER). Brown Norwegian rat leukaemia (BNML) was used as an in vivo model system. 8-CPT-cAMP- and TTAs anti-leukemic activity was assessed in vivo and in vitro. The compounds were able to synergistically induce a decrease in viability in promyelocytic IPC-81 cells when measured by combinational index. Enforced expression of ICER could partially protect the cell from TTA induced apoptosis. Bcl-2 over expression did not prevent additive apoptosis induction of the two compounds. An additive proliferation inhibition of the two compounds was detected in HL-60 cells at a 8-CPT-cAMP concentration that did not affect viability in itself. TTA and 8-CPT-cAMP had a spleen anti-leukaemic activity in vivo but the schedule of administration will have to be further investigated. In conclusion there seems to be a converging point in the action of 8-CPT-cAMP and TTA since they synergistically decrease in vitro viability of acute leukemic cells.
Pharmacokinetic and Pharmacodynamic Properties of SB1317, a Potent and Orally Active Multi-Kinase Inhibitor.
Authors: Yeo Pauline , Venkatesh P. , Goh Evelyn , Zeng Peizi , New Lee S. , Hu Changyong , Sangthongpitag Kanda , William Anthony D. , Goh Kee C. , Wood Jeanette M. , Kantharaj Ethirajulu
Published: October 2019

Key physicochemical properties such as solubility, lipophilicity (logD7.4, logP) and pKa (the negative log of the acid dissociation constant) can be used to predict protein binding, tissue distribution, and gastrointestinal (GI) absorption. More recent application of computational methods allows even better prediction of compound oral bioavailability from in vitro and/or in silico properties. During the lead optimization process in our multi-kinase inhibitor program a good correlation between physicochemical properties like logD and solubility (determined experimentally at pH 7) versus AUC(0-inf) (Area Under the Curve) was observed for a series of kinase inhibitors. A calculated logD*solubility value between 200 to 400 predicted a reasonably high oral AUC(0-inf) in mouse PharmacoKinetic (PK) studies for this series of compounds, with correlation coefficient of >0.9. The correlation of logD*solubility vs. AUC(0-inf) enabled us to prioritize compounds for PK studies. SB1317, a novel pyrimidine derivative, is an orally active multi-kinase inhibitor that evolved as a lead drug candidate from in vitro and in vivo pharmacokinetic studies. SB1317 is a highly permeable compound, does not undergo active P-glycoprotein transport, and has a solubility of 75 μg/ml. It is metabolically stable in dog and human liver microsomes and unstable in rodent liver microsomes. No P450 inhibition was observed up to 10 μM towards CYP3A4, CYP1A2, CYP2C9 and CYP2C19. In vivo pharmacokinetics in nude mice and Beagle dogs resulted in %F of 12 and 37% respectively. Tumor pharmacokinetics of SB1317 shows increased exposure in tumor than in plasma with a AUC(0-t) tumor/plasma ratio of 3. Excellent oral dose proportionality (10, 20 and 40mg/kg) was observed in both plasma and tumor. SB1317 has exhibited uniformly high plasma protein binding (>99%) across the preclinical species and human. It is the free or unbound portion of the compound in the plasma or at the tissue level that would yield effective anti-tumor activity. (AUC0-inf)unbound/GI50 and Cmax-unbound/GI50 were used as PK/PD surrogates for the measure of SB1317 efficacy. A factor of SB1317 unbound AUC (0-inf)/GI50 above 0.5 results in significant pharmacodynamic (PD) effects in hematological tumor models (MV4-11 and HL-60 engraft model). Linear pharmacokinetic extrapolation from preclinical species to human was determined by allometric scaling (predicted human % F = 36). PK/PD relationships established for SB1317 in preclinical species could form the basis of a PK/PD-driven clinical development program.
Iv Vitro Modeling of the Use of FLT3 Inhibitors To Reduce Chemotherapy Doses for Elderly Patients with AML.
Authors: Schittenhelm Marcus M. , Kampa Kerstin M. , Yee Kevin W.H. , Kanz Lothar , Heinrich Michael C.
Published: October 2019

Among the expanding population of elderly AML patients, aggressive therapy options are often limited due to other medical conditions and the decreased tolerance of these patients to conventional chemotherapy. Internal tandem duplications (ITD) of the FLT3 juxtamembrane domain occur in 20–30% of AML patients and predict poor outcome. Monotherapy with FLT3-tyrosine kinase inhibitors (TKIs) has produced biological and some clinical responses. Side effects of TKI and chemoagents are only moderately overlapping. We hypothesized that the use of FLT3 inhibitors may allow dose reduction of chemotherapy with resultant decreased side effects, but without loss of antileukemic activity. Using primary AML blasts, we found that combination of the TKI tandutinib (Tn) (or sunitinib (Sn)) with cytarabine (Cy) and/or daunorubicin (Dn) has a favorable growth inhibitory activity compared to Cy/Dn alone. Of note, the effects were only seen in cells expressing a FLT3 ITD mutation. In order to perform more detailed mathematical analyses, we utilized three cell lines containing FLT3 ITD mutations: a BaF3 cell line transduced with an ITD mutant FLT3 as well as MV4-11 and MOLM14 cell lines which have naturally occurring FLT3 ITD mutations. Dilution series were set up with the single agents as well as different drug combinations to create isobolograms using the method of Chou and Talalay. Next, dose reduction indices (DRI) were computed using calcusyn® software. DRI for the combination of Tn with Cy demonstrated a 6 (MV4:11) - 19 (Ba/F3 FLT3 ITD) fold potential dose reduction of Cy at ED90 for induction of apoptosis. In addition, the DRI of cytarabine in combination with Sn would allow a 2–145 fold cytarabine dose reduction for the various cell lines. The combination of Dn with Tn or Sn demonstrated a DRI up to 6 fold. Similar DRI were seen when inhibition of cellular proliferation was the final endpoint. Of note, Sn plus Cn or Dn displayed a - moderate but beneficial - DRI of 2 in Ba/F3 cells expressing the FLT3 wildtype isoform. Also, triple-combination allowed up to 10 fold dose reduction of the chemoagents - in cell lines as well as native blasts. We conclude that dose reduction strategies by adding TKI may be clinically useful, particularly for elderly AML patients or patients with co-existent illnesses that would predict for a poor tolerance of standard chemotherapy regimens.
Expression Analysis and Prognostic Significance of the PHLPP Gene in Acute Leukemias and Myelodysplastic Syndromes.
Authors: Ono Takaaki , Nakamura Satoki , Kazuyuki Shigeno , Takeshita Kaori , Keiji Okinaka , Hirano Isao , Fujisawa Shinya , Ohnishi Kazunori
Published: October 2019

[Background] The proto-oncogene Akt/PKB is activated in many human cancers including leukemias, and regulates the cell survival and apoptosis. Akt/PKB is activated by growth factors or the phosphatidylinositol-3,4,5-triphosphate via PI3K, phosphorylates many substrates for cell survival or proliferation. The tumor suppressor PTEN prevents Akt/PKB phosphorylation and activation. On the other hand, the PH domain leucine-rich repeat protein phosphatase (PHLPP) dephosphorylates Akt/PKB, induces apoptosis, and suppresses tumor growth. It has been reported that the expression levels of PHLPP are significantly reduced in several colon cancer and glioblastoma cell lines, and Akt/PKB phosphorylation is elevated. However, we found that PHLPP gene was overexpressed in MNCs derived from patients with acute leukemias and MDS. In this study, we have investigated the expression analysis and prognostic significance of the PHLPP gene in patients with acute leukemias and MDS.

[Methods] The cells used in this study were human acute leukemia cell lines, HL60, U937, YRK2, CEM, and MOLT4 cells. Primary acute lymphoblastic leukemia (8 ALL (8 L2)), myelodysplastic syndromes (32 MDS (18 RAEB-I, 14 REAB-II)), and acute myeloblastic (25 AML (4 M1, 11 M2, 6 M4, 4 M5)) cells were obtained from the peripheral blood. Human normal mononuclear cells (MNCs) were isolated from peripheral blood (PB) of healthy volunteers after obtaining informed consents. For analysis of PHLPP mRNA, quantitative RT-PCR was performed in all cell lines and clinical samples.

[Results] In all cell lines, the expressions of PHLPP mRNA were significantly increased more than normal MNCs. In HL60, U937, YRK2, CEM and MOLT4 cells transfected with the siRNA PHLPP, it is shown that the phosphorylation state of Akt was markedly increased compared to the untransfected cells. In the clinical samples obtained from patients with acute leukemias and myelodisplastic syndromes, the PHLPP gene was overexpressed in 7/8 (87.5%) of ALL, 14/25 (56%) of AML, and 26/32 (81.3%) of MDS. This higher expression was not associated to the counts of blasts of PB (P < 0.01). The relative folds of PHLPP gene expression were for AML: 3.02, MDS: 4.16, and ALL 3.62 compared to normal MNCs. Kaplan-Meier survival analysis revealed that lower PHLPP expression increases the probability of the longer overall and the progression free survival of the patients. Moreover, when CR status after chemotherapy, mean PHLPP expression levels were significantly reduced compared to unCR status after chemotherapy in MNCs derived from PB of patients with acute leukemias and myelodysplastic syndromes.

[Conclusions] Our results suggest that the PHLPP gene was overexpressed in MNCs derived from the patients with acute leukemias and myelodysplastic syndromes and may possibly be characterized as a new marker of unfavorable prognosis for ALL, AML, and MDS.
Telomerase Activity and Telomere Length in APL: May Be Proliferative and Prognostic Markers.
Authors: Ghaffari Seyed Hamidolah , Shayan-Asl Nilofar , Jamialahmadi Abdolhamid , Ghavamzadeh Ardeshir , Alimoghaddam Kamran
Published: October 2019

Introduction: The telomeric DNA together with its associated proteins protects the chromosome ends from degradation or aberrant recombination. Telomerase and telomere are extensively investigated as potential diagnostic and prognostic markers in human tumors. In this study, we aim to investigate the significance of telomerase activity (TA) and telomere length (TL) in patients with acute promyelocytic leukemia (APL).

Methods: About 300 sequential peripheral blood mononuclear cell (PBMC) samples were collected from 40 patients with APL (32 newly diagnosed and 8 relapsed), at diagnosis, during and after therapy with Arsenic Trioxide. TA was assessed by TRAP-ELISA and -PAGE procedures. Terminal restriction fragment (TRF) length was determined by Southern blot analysis, using a Chemiluminescence-based assay. Quantification of PML-RARα/G6PDH transcripts was carried out by real-time PCR assay.

Results: About 90% of the APL patients had a significant reduction in TRF length (median 3.5, ranged 2.3 to 6.7 kbp) relative to the age-matched control or to that at the time of CR (median 11.37; ranged 8.90 to 14.70 kbp) from the same patients (P<0.0001). A significance positive correlation between telomere length and PML-RARα expression was found in the APL patients (P=0.001). In all the APL patients, telomerase activity was elevated in comparison with normal individuals (P<0.001). The telomerase activity levels were significantly higher in patients with short TRF. The group of patients with shortened TRF and elevated TA had a significantly poorer overall survival. The levels of TA were even significantly higher and the TL size difference of leukemic from non-leukemic was significantly longer in relapsed patients than patients with the primary APL.

Conclusion: Short telomere and high telomerase levels in the APL patients are mainly a consequence of extensive proliferative histories and they correlate with the disease progression. The shortened telomere length and the elevated telomerase activity may serve as prognostic factors for a subset of APL patients with more aggressive disease and poor outcome, and who may not respond favourably to arsenic therapy.