bluuebunny/crossref_metadata_2025 huggingface dataset:
Dual Role of Pim-2 Depends on the Associated Proteome.
Authors: Levy Daphna , Cohen Amos M. , Don Jeremy
Published: April 2020

Pim-2, a Ser/Thr kinase, is a proto-oncogene originally identified as common proviral insertion sites in T and B cell lymphomas in mice. Deregulation of Pim-2 expression has been documented in several human malignancies, including leukemia, lymphoma, multiple myeloma and prostate cancer. In human non-Hodgkin’s lymphomas and in chronic lymphocytic leukemia Pim-2 is up-regulated and its expression correlates with disease activity. Pim-2 promotes cell survival in response to a wide variety of proliferative signals. Pim-2 promotes cell survival by phosphorylation of Bad and Cot. The goal of this study was to clarify the significance of our new unanticipated aspect of Pim-2′s function, namely, that over-expression of Pim-2 in HeLa cells led to cell cycle arrest at G1 and to increased apoptosis. We found that the G1 arrest was associated with increased (T14/Y15) phosphorylation of CDK2, increased proteosomic degradation of CDC25A, and increased levels of the CDK inhibitor p57. In addition, we found increased E2F-1 levels, which suggested the usage of the E2F-1 dependent apoptotic pathway. Using dominant negative forms of either E2F-1 or p73, which were co-expressed with Pim-2 in HeLa cells, revealed significant rescue of the G1 arrest and apoptotic phenomena. Silencing of Pim-2 in these cells, via siRNA, reversed the G1 arrest and pro-apoptotic effects, and verified the Pim-2 dependent specificity. We conclude that Pim-2 might play a dual role. Our data suggest that under certain environmental circumstances and in various cell types, Pim-2 appears to increase cell survival by abrogating some pro-apoptotic substrates, but under different proteomic associations Pim-2 might favor G1 arrest and apoptosis.
Computational Prediction of c-MYC Binding and Action by Integration of Multiple Data Sources.
Authors: Chen Yili , Blackwell Thomas W. , Gao Jing , Hewagama Anura , Grifka Heather M. , Lee Angel W. , States David J.
Published: April 2020

c-MYC is an important proto-oncogene. Its actions are mediated by sequence specific binding of the c-MYC protein to genomic DNA. While many c-MYC recognition sites can be identified in c-MYC responsive genes, many others are associated with genes showing no c-MYC response. It is not yet known how the cell determines which of the many c-MYC recognition sites are biologically active and directly bind c-MYC protein to regulate gene expression. We have developed a computational model that predict c-MYC binding and functional activation as distinct processes. Our model integrates four types of evidence to predict functional c-MYC targets: genomic sequence, MYC binding, gene expression and gene function annotations. First, a Bayesian network classifier is used to predict c-MYC recognition sites likely to exhibit high occupancy binding in chromatin immunoprecipitation studies using several types of sequence information, including predicted DNA methylation using a computational model to estimate the likelihood of genomic DNA methylation. In the second step, the DNA binding probability of MYC is combined with the gene expression information from 9 independent microarray datasets in multiple tissues and the gene function annotations in Gene Ontology to predict the c-MYC targets. The prediction results were compared with the c-MYC targets in public MYC target database [www.myccancergene.org], which collected the c-MYC targets identified in biomedical literatures. In total, we predicted 599 likely c-MYC genes on human genome, of which 73 have been reported to be both bound and regulated by MYC, 83 are bound by MYC in vivo and another 93 are MYC regulated. The approach thus successfully identified many known c-MYC targets as well as suggesting many novel sites including many sites that are remote from the transcription start site. Our findings suggest that to identify c-MYC genomic targets, any study based on single high throughput dataset is likely to be insufficient. Using multiple gene expression datasets helps to improve the sensitivity and integration of different data sources helps to improve the specificity.

Summary of c-MYC Targets Prediction Microarray Dataset Data Source (Citation) Tissue Predicted Targets Binding&Regulation Reported Only Binding Reported Only Regulation Reported 1 PMID: 15778709 B Cell 421 61 60 56 2 PMID: 12086878 Prostate Cancer 428 56 65 76 3 PMID: 14722351 Prostate Cancer 50 4 7 13 4 PMID: 15254046 Prostate Cancer 66 19 8 14 5 PMID: 12747878 Breast Cancer 17 1 3 5 6 PMID: 11707567 Lung Cancer 295 51 42 59 7 PMID: 15820940 CML 8 1 1 2 8 PMID: 12704389 ALL 222 45 32 46 9 PMID: 11731795 ALL / MLL / AML 22 6 1 6 Total 599 73 83 93
Transcriptional Regulation of the SHP-1, a Candidate Tumor Suppressor Gene in Hematological Malignancies.
Authors: Nakase Koichi , Cheng Jihua , Zhu Quan , Marasco Wayne A.
Published: April 2020

The SH2-Containing Protein-Tyrosine Phosphatase-1 (SHP-1) is a hematopoietic cell specific protein-tyrosine phosphatase and a candidate tumor suppressor. It has been shown that expression of SHP-1 is repressed in HTLV-1 transformed lymphocyte cell lines, Adult T-cell Leukemia and other hematological malignancies. However, the mechanisms that regulate SHP-1 expression remain unclear. We previously identified that the −120 to +157 of the SHP-1 P2 promoter (core promoter) exhibits similar levels of activity as the wild type promoter. Here we report the involvement of NF-kB and Oct-1 in the transcriptional regulation of the SHP-1 P2 promoter. To elucidate the mechanisms by which SHP-1 promoter is regulated, in vitro DNase I footprinting experiments were performed. Incubation of nuclear extracts from Jurkat cells with the SHP-1 P2 promoter probes revealed two DNase I protected regions between −100 and −90 and between −80 and −55 in the SHP-1 P2 promoter. Elk-1, Ik-1, NF-kB and Oct-1 were identified through database search as likely candidate factors binding to these regions. Electrophoretic Mobility Shift Assay (EMSA) was carried out using a double strand DNA probe that covers the previous DNase I footprinting area (−120 to −19) and Jurkat nuclear extract. Transcription factors NF-kB and Oct-1 were confirmed as binding to the SHP-1 P2 promoter through antibody supershift, probe competition, and/or site directed mutagenesis. Results from in vivo Chromatin Immunoprecipitation (ChIP) assay with anti-NF-kB and anti-Oct-1 antibody also strongly suggested that Oct-1 and NF-kB bound the SHP-1 P2 promoter in Jurkat cells. In transient transfection experiments using luciferase as a reporter, mutations at Oct-1 binding sites and NF-kB binding sites showed significant reduction in SHP-1 promoter activity compared to wild type core promoter, further indicating the involvement of these factors in the SHP-1 P2 promoter transcription regulation. In summary, this is the first report demonstrating that Oct-1 and NF-kB are involved in the regulation of SHP-1 P2 promoter. Modulation of these transcription factors could provide new targets for treatment of hematological malignancies.
ERBB Signaling Promotes BCR-Abl Expression and Modulates Multiple Signaling Pathways as Assessed by High-Throughput, Reverse Phase Protein Array.
Authors: Nelson Laura D. , Siwak Doris R. , Lu Yiling , Miller Claudia , Chandra Joya , Hughes Dennis P.M.
Published: April 2020

ERBB family receptor tyrosine kinases, EGFR, Her-2, Her-3 and Her-4, are prognostic and therapeutic targets in multiple carcinomas, including breast, lung, and head and neck tumors. We previously reported aberrant expression and activation of Her-2 and Her-4 in a panel of Ph+ ALL cell lines: Z33, Z119, and Z181. Loss of cell viability and apoptotic DNA fragmentation were observed after treatment with the irreversible pan ERBB inhibitor, CI-1033, in vitro. In the present study, we define the mechanisms of cell death initiated by ERBB blockade. We examined the expression and Tyr1248 phosphorylation of Her-2 in Z119 and Z181 cell lines upon CI-1033 treatment and found a 4-fold decrease in Ph-Y1248 by Western blotting. We then assessed the impact upon downstream signaling pathways using reverse-phase protein microarray (RPPA) - a new high-throughput method to measure and profile the total expression and post-translational phosphorylation of signaling pathway components. Leukemia cells treated with CI-1033 assessed with RPPA using 34 different antibodies showed mTOR expression to be decreased by more than two-fold in Z119 and Z181. Concomitantly, we saw a more than three-fold decrease in phosphorylated S6-S240/244. This suggests that down-regulation of translational control caused by inhibition of activated ERBB-family receptors contributes to the reduced proliferation in these cells. We also observed a 3.5-fold decrease in phosphorylated glycogen synthase kinase (GSK)-3a/b 21/9 in Z119 and a 1.7-fold decrease in Z181. This suggests inactivation of GSK3, which is regulated by Akt/PKB signaling. We also observed a four-fold decrease in Bcr-Abl expression in Z181, identified as p190 by Western. This suggests that ERBB signaling helps maintain expression of Bcr-Abl protein, driving Ph+ lymphomagenesis, perhaps explaining why ERBB blockade induces apoptosis in Ph+ ALL. Elucidating the mechanism of this apoptosis, CI-1033 caused activation of caspase-3 in Z119 and Z181 as measured by DEVD-AMC cleavage, and caused PARP cleavage. Thus multiple, non-redundant signaling pathways downstream of ERBB can be downregulated with a small molecule inhibitor, CI-1033, causing reduced viability and increased apoptosis in ERBB+ Ph+ ALL cell lines. Our data suggest that inhibition of these signals may provide additional therapeutic efficacy to Ph+ ALL patients.

Figure Figure
Single Versus Double Umbilical Cord Blood Transplantation (UCBT): Higher Risk of Acute Graft-Versus-Host Disease (GVHD) but Lower Transplant Related Mortality (TRM) in Recipients of Double UCBT.
Authors: MacMillan Margaret L. , Brunstein Claudio , DeFor Todd E. , Blazar Bruce R. , Weisdorf Daniel J. , Wagner John E.
Published: April 2020

Transplantation of 2 partially HLA matched UCB units has been shown to be a safe and effective means to overcome the cell-dose barrier in adolescents and adults. In addition, preliminary data suggest that double UCBT is associated with a greater graft-versus-leukemia effect (Blood2005:106:93a). To date, there has been no comparative analysis on the risk of acute GVHD in this population of patients. Therefore, we compared rates of acute GVHD in recipients of single (n=210) versus double (n=169) UCBT. Double UCB recipients were more likely to be older, transplanted in recent years, treated with a non myeloablative therapy, received mycophenolate mofetil as GVHD prophylaxis, and less likely to receive antithymocyte globulin (ATG) as part of the conditioning. Incidences of grades II–IV and III–IV acute GVHD were higher in recipients of double than single UCBT recipients (60% vs 33%, p.01) and (21% vs 11%, p=.01), respectively. Adjusting for differences between groups, two factors were associated with the development of grade II–IV acute GVHD in Cox regression: use of 2 UCB units (RR 2.0 vs 1.0 [95% CI, 1.3–3.2, p.01]) and absence of ATG in the preparative regimen (RR 1.0 vs 0.5 [95% CI, 0.3–0.8, p.01]). Other factors that were tested in the model but not determined to be significantly associated with acute GVHD were recipient age, gender, weight, diagnosis, time from diagnosis to UCBT, HLA disparity, total and CD3 cell dose, CMV serostatus, conditioning regimen, and GVHD prophylaxis. Despite increased risk of acute GVHD, TRM 1 year was significantly lower in recipients of double UCBT (17%, 95% CI, 5–29%) as compared to recipients of a single UCBT (47%, 95% CI, 26–68%; p =.02) (Figure 1) among those that developed grade III–IV acute GVHD. Survival at 1 year among those with grade III–IV acute GVHD was significantly higher after double UCBT (67%, 95% CI, 51–83%) than after single UCBT (41%, 95% CI 21–61%; p=.04) (Figure 2).

Figure 1 Figure 1. Figure 2 Figure 2.

In conclusion, risk of acute GVHD is significantly higher in recipients of two partially HLA matched UCB units, yet it does not appear to adversely effect TRM or survival. ATG in the preparative therapy, however, does not appear to reduce TRM despite its favorable association with reduced GVHD. Impact of GVHD on immune recovery and infection risk in recipients of single and double UCBT is under investigation.
Inhibition of UBE2Q2 Sensitizes Cells to Mitotic Arrest and Cytotoxicity Caused by Vincristine.
Authors: Crawford David F. , Banerjee Sami , Brooks William S.
Published: April 2020

A microarray study was previously performed to identify G2/M-specific genes and genes with altered expression in response to G2 DNA damage checkpoint activation. We predicted that many of these proteins would be mitotic regulatory proteins which might serve as potential targets in the treatment of malignancies. Included in this set were several proteins involved in ubiquitin-mediated protein degradation including a putative ubiquitin conjugating enzyme known as UBE2Q2. We have demonstrated that this gene is expressed in a cell cycle dependent manner and that the encoded protein can function as an ubiquitin conjugating enzyme (or E2) in vitro. To assess the role of this protein in mitosis and the cellular response to treatment with chemotherapy agents, we inhibited UBE2Q2 function by expressing a dominant-negative version of the protein. With dominant-negative UBE2Q2 (DN-UBE2Q2) expression, no alteration in normal cell cycle kinetics is observed in unperturbed cells. However, DN-UBE2Q2 expression causes a dramatic change in the cellular response to vincristine and other microtubule inhibiting agents (MIAs). While control cells transiently arrest in mitosis after low dose vincristine treatment, DN-UBE2Q2-expressing cells have an impaired ability to escape this arrest which leads to a dramatic potentiation of the mitotic arrest. Cells expressing DN-UBE2Q2 are also much more sensitive to the cytotoxic effects of both vincristine and paclitaxel, MIAs with distinct mechanisms of action. Although DN-UBE2Q2 causes sensitization to all MIAs tested, it does not confer increased sensitivity to etoposide or doxorubicin, two DNA damaging agents, indicating that the effect is specific for microtubule disruption. The increased sensitivity to MIAs conferred by DN-UBE2Q2 expression is a result of caspase-mediated apoptosis and can be blocked using a caspase inhibitor. In order to independently confirm these findings, we also inhibited UBE2Q2 expression using siRNA. As with dominant-negative expression, we found that RNAi directed against UBE2Q2 enhanced the vincristine-induced mitotic arrest. These findings suggest that inhibition of UBE2Q2 might be used to augment the effectiveness of MIAs in the treatment of hematologic and other malignancies.
Pleiotrophin Promotes Myeloma Proliferation through the Activation of Known and Novel Receptors.
Authors: Gordon Melinda S. , Lee Hee Jin , Campbell Richard A. , Drucker Charles B. , Katz Matthew , Leuchter Richard K. , Bonavida Benjamin , Berenson James R.
Published: April 2020

Myeloma survival and proliferation in the bone marrow (BM) depends on the expression of a variety of autocrine and paracrine growth factors, including IL-6, insulin-like growth factor I (IGF-I), vascular endothelial growth factor (VEGF), and hepatocyte growth factor (HGF). We recently discovered an additional new autocrine myeloma growth factor, pleiotrophin (PTN). PTN is an 18kD heparin-binding protein normally expressed during early development and downregulated in adults, but aberrant PTN re-expression has been associated with a variety of aggressive solid tumors including neuroblastoma, glioma, melanoma and lung, breast and prostate cancers. We found that interference with PTN using a polyclonal anti-PTN antibody inhibited the proliferation of myeloma cells in vitro and in vivo by inducing cell cycle arrest but not apoptosis. To determine the mechanism by which PTN stimulates myeloma proliferation we analyzed the expression of the known PTN receptors, syndecans 1 (CD138) and 3, the anaplastic lymphoma kinase (ALK) and the receptor tyrosine phosphatase beta/zeta (RPTPβ/ζ) on myeloma cells. The expression of ALK and RPTPβ/ζ has not previously been investigated in any hematologic malignancy. In addition to syndecan 1 we found that a subset of myeloma cell lines and BM mononuclear cells (BMMCs) from myeloma patients are RPTPβ/ζ+ by RT-PCR, Western blot and flow cytometry. Myeloma cells, however, do not express ALK. RPTPβ/ζ inhibition by PTN binding leads to the accumulation of β-catenin, and downstream activation of Wnt, NF-κB, MAPK and Akt-mediated cell signals in cells from solid tumors, signaling pathways known to contribute to myeloma cell survival and proliferation. The myeloma cell line MM-1S and the SCID-hu myeloma model LAGλ-1 are both CD138+ but RPTPβ/ζ−. Nonetheless, the growth of these cells is inhibited by anti-PTN antibody. CD138 lacks cytoplasmic signaling motifs suggesting that additional novel receptors are required for PTN-stimulated cell growth in these cells. To continue to define PTN-mediated signaling in myeloma cells we compared the induction of PTN-regulated signaling pathways in RPTPβ/ζ+ RPMI 8226 cells compared to RPTPβ/ζ − MM-1s cells by phospho-protein Western blot. We found differences in PTN-stimulated tyrosine phosphorylation between RPTPβ/ζ+ and RPTPβ/ζ −cells. Specifically, we also discovered that RPTPβ/ζ+ RPMI 8226 cells activate the MAPK Erk1/2. In contrast, we found Akt, but not Erk1/2, to be activated by PTN in RPTPβ/ζ − MM-1S cells. We are now continuing our analysis of PTN signaling in these cells. We are using also identifying new PTN receptors on the surface of myeloma cells by affinity chromatography using biotinylated-PTN. These studies will provide potential new targets that should lead to the development of novel targeted anti-myeloma therapies.
Effect of Cyclosporine A and Estrogen-Receptor Inhibitor on the Reversion of Multidrug Resistance of K562/A02 Line.
Authors: Chen Bao-An , Bao Wen , Gao Feng , Cheng Jian , Gao Chong , Ding Jia-Hua , Sun Yun-Yu , Wang Jun , Zhao Gang , Chen Ning-Na , Fei Fei
Published: April 2020

Objective: This paper was to study the reverse effect of cyclosporine A and the estrogen-receptor inhibitor, raloxifene, on multidrug resistance cell line K562/A02 and to investigate the reversal mechanism of this combination, and to provide theoretic evidence for the clinical application of them as resistance modifying agents.

Method: The IC50 (the concentration causing 50% inhibition of cell growth) of DNR were assayed by MTT method; mdr-1 mRNA was assayed by semi-quantitative reverse transcriptase polymerase chain reaction (RT-PCR).; intracellular DNR concentrations and the apoptosis and p-glycoprotein (p-gp) expression in K562 and K562/A02 were detected by fluorometry .

Results:

The IC50 of DNR for K562/A02 and K562 cells were 23.51mg/L and 0.29mg/L respectively. Pretreating K562/A02 cells with CsA(1mg/L) or raloxifene(2.5mg/L) for 48 hours partially restored the sensitivity of K562/A02 cells to DNR (IC50 were5.98mg/L and 8.15mg/L respectively) ,but had no effect on K562 cells;IC50 of combined cyclosporine A and raloxifene was 3.68mg/L The intracellular DNR concentrations( [DNR]i) in K562 cells were higher than that in K562/A02 cells,[DNR]i in K562/A02 cells was 12% of K562. Pretreating K562/A02 cells with cyclosporine A and raloxifene alone or combination for 48 hours induced the enhancement of [DNR]i in K562/A02 cells([DNR]i:CsA alone 33% of K562; raloxifene alone 12.78%of K562;CsA+raloxifene 45.29% of K562 Cyclosporine A and raloxifene alone or combination could decrease the expression of P-gp in K562/A02,the effect for 48 hours as follows: control(K562) 3.58,control(K562/A02) 104.96; CsA alone 78.29; raloxifene alone 85.02;CsA+raloxifene 59.89 K562/A02 showed apoptotic characteristics after treated with cyclosporine A for 48 hours and no apoptotic characteristics after treated with raloxifene for 48 hours Cyclosporine A and raloxifene alone could down regulate the expression of mdr-1 gene mRNA in K562/A02, cyclosporine A and raloxifene combination could markedly down regulate the expression of mdr-1 gene mRNA in K562/A02, the effect for 48 hours as follows(mdr1/β-action):control 1.313;CsA alone 1.232;raloxifene alone 1.052;CsA+raloxifene 0.449.

Data was analyzed by SPSS 11.0 software and expressed as mean ± SD.

Conclusions:

Activation of P-gp may be involved in the mechanism of MDR of K562/A02 cell line; Multidrug resistance (MDR) can be partially reversed by CsA or raloxifene, of which the combination shows a great synergistic reversal effect.
A Farnesyltransferase Inhibitor Manumycin A Induces Oxidative DNA Damage and Apoptosis Via Oxidative Stress in Leukemia Cells.
Authors: She Miaorong , Kunyuan Guo , Hu Haiyan , Niu Xinqing , Tu Sanfang , Lu Xiaoxun
Published: April 2020

Farnesyltransferase inhibitors (FTIs) are currently under investigation for the treatment of leukemia. The mechanism of action of FTIs has not been clarified .In this study, we investigated the mechanism of actions of FTI manumycin in acute myeloid leukemia (AML) cells. We found that manumycin A (Manu) had an antineoplastic and apoptotic effect on leukemia cells. Manu significantly increased 8-OhdG, a reliable marker for assessment of oxidative DNA damage, formation. Manu also rapidly increased the phophorylation of H2AX in HL-60 and U937 cells. Methoxyamine, an inhibitor of base excision repair, enhanced the phophorylation of H2AX induced by Manu. Furthermore, Manu could induce phosphated p38 MAPK in HL-60 and U937 cells, and inhibitor of p38 MAPK could rescue leukemia cells from Manu-induced cytotoxicity. Moreover, Quenching of ROS with NAC prevented DNA damage, p38 phosphorylation and apoptosis by Manu. These results suggest the hierarchical position of ROS in oxidative DNA damage and apoptosis pathway. In conclusion, FTI induced oxidative DNA damage by induction of ROS and initiated the DNA damage responses then trigger the apoptosis involved p38 MAPK activation.
Autophagy and Apoptosis Are Induced Simultaneously in Leukemia Cells by Vitamin K2.
Authors: Miyazawa Keisuke , Yokoyama Tomohisa , Toyotake Juri , Gotoh Akihiko , Naito Munekazu , Ito Masahiro , Yuo Akira , Kondo Seiji , Ohyashiki Kazuma
Published: April 2020

Autophagy is a membrane trafficking process that leads to degradation of cytosolic proteins and organelles by lysosomes, and it is conserved throughout evolution. Autophagy is induced above basal levels in response to nutrient deprivation or trophic factor withdrawal, and it sustains metabolism through the targeted catabolism of long-lived proteins. Thus, autophagy acts as a self-limited survival mechanism. However, recent findings have suggested that autophagy is also as a potential contributor to programmed cell death.

We previously reported that vitamin K2 (VK2) selectively and potently induces the apoptosis of cultured acute myeloid leukemia cells (AML) and AML cell lines, but has almost no effect on normal hematopoietic cells. In addition, the clinical benefit of using VK2 for patients with AML and myelodysplastic syndrome has been reported. In the present study, we focused on whether autophagy was induced in AML cells by treatment with VK2.

HL-60bcl-2 cells with stable transfection of the bcl-2 gene showed 5-fold greater expression of BCL-2 compared with HL-60neo cells, and these cells were resistant to apoptosis induced by VK2, along with the occurrence of monocytic differentiation via G1 arrest. This suggested that VK2 had a differentiation-inducing effect on AML cells that were resistant to apoptosis. However, a cytocidal effect was still detectable in a caspase-independent manner. Treatment of HL-60bcl-2 cells with VK2 resulted in an increase of acid vesicular organelles (AVO) detected by acridine orange staining (for lysosomes).

Immunonoblotting with an antibody for microtubule-associated protein-1 light chain-3 (LC3) revealed the conversion of LC3-I into LC3-II a form that migrates more rapidly on SDS-PAGE and participates in the formation of autophagosomes (one of the hallmarks of autophagy). Electron microscopy revealed an increase of autophagosome formation after exposure to VK2. Finally, the induction of caspase-independent cell death by VK2 was suppressed in the presence of 3-methyladenine. These results suggest that autophagic death of HL-60bcl-2 cells was induced by VK2. Furthermore, detailed electron microscopic assessment of HL-60neo cells during exposure to VK2 revealed that autophagosome formation was more prominent compared with that occurring in H-60bcl-2 cells, although nuclear chromatin condensation was also observed at the same time. These findings indicated a mixture of the morphologic features of apoptosis and autophagy. The present results suggest that autophagy and apoptosis can both be induced simultaneously by VK2. However, autophagy only became detectable when cells were protected from rapid apoptotic death.
Polydeoxyribonucleotides Antagonize Nucleoside Analogues in Cancer Therapy.
Authors: Yang Ting , Steinmann Ingo , Tank Simone , Chen Zhizhe , Kolb Hans-Jochem , Buhmann Raymund
Published: April 2020

Background: In the past years, an increasing number of nucleic acid based drug candidates (e.g. antisense oligonucleotides, aptamers, ribozymes, RNA interference) have been identified and already tested in clinical trials. But until now, no information is available whether these treatment approaches might interfere with chemically related drugs, e.g. nucleoside analogues used in cancer or viral therapy.

Objective: In the current survey we investigated, whether randomly synthesized single-stranded polydeoxyribonucleotides of different length could interfere with the cytotoxic effects of nucleoside analogues (e.g. fludarabine, cytarabine) on lymphocytes or myeloid blasts.

Methods: For this purpose, T-lymphocytes or CD33 positive myeloid blasts (purity >95%) were labelled with [1μM] carboxyfluorescein diacetate, succinimidyl ester (5(6)-CFDA-SE) and incubated with different concentrations of fludarabine or cytarabine (200μM, 20μM und 2μM). Cellular proliferation was induced by addition of CD3/CD28 Dynabeads for the T-cells, or a cytokine cocktail containing 50ng/mL of SCF, 25ng/mL of IL-3, 100ng/mL of GM-CSF, 100ng/mL of G-CSF, 2 U/mL of EPO, 0.47g/L of transferrin, and 5×10-5mmol/L of 2-ME for the myeloid blasts. Random polydeoxribonucleotides of different length (20–80b) and concentrations were added immediately or with a delay of 24, 48 or 72 hours to the assay. After 5 days, quantitative CFDA distribution was measured by flow cytometry to assess the cellular proliferation of the T cells and the myeloid blasts. Moreover, cell viability was measured by trypan blue exclusion. Each experiment was performed at least three times.

Results: Polydeoxyribonucleotides of different length and composition could antagonize the cytotoxic effects of nucleoside analogues (fludarabine, cytarabine) in both, T-cells and myeloid blasts. Thereby, the antagonistic effects of polydeoxyribonucleotides revealed to be concentration- and time dependent. Moreover interference with a equimolar concentrations of polydeoxyribonucletides could be detected even with a delay of 48 hours. Of importance, the concentration of nucleoside analogues used in these experiments, referred to concentrations applied in standard clinical treatment protocols.

Conclusion: Treatment approaches using polydeoxyribonucleotides, might be critical in context with nucleoside analogues, like fludarabine and cytarabine and diminish the efficacy of these drugs. Further experiments are necessary to elucidate the precise mechanism of these effects.
Hypoxia Induces Imatinib Resistance in CML Cell Lines.
Authors: Buglio Daniela , La Cava Piera , Stagno Fabio , Chiarenza Annalisa , Berretta Salvatore , Aloisi Alesandra , Palumbo Giuseppe A. , Vigneri Paolo , Di Raimondo Francesco
Published: April 2020

Background: Imatinib is a potent tyrosine kinase inhibitor that is highly effective in the treatment of chronic myeloid leukemia. However, some patients are resistant to imatinib. Some of the mechanisms leading to imatinib resistance include amplification of the BCR-ABL gene, determining overexpression of the protein, and mutations in the BCR-ABL protein with alteration of imatinib binding sites. Imatinib uptake is an active process mediated by a group of transporters that includes the organic cation transporters (hOCT) and it has been shown that different expression of OCT1 may play a critical role on intracellular drug levels and, hence, resistance to imatinib. Hypoxia is another important factor that may contribute to drug resistance. Hypoxia-Inducible Factor (HIF-1α) and its downstream target, Vascular endothelial Growth Factor (VEGF), have been shown to be overexpressed in leukemic bone marrow specimens compared to normal bone marrow. The purpose of this study is to determine if the hypoxic conditions and OCT1 inhibition affect imatinib sensitivity.

Methods: Chronic myeloid leukemic cell line K562 and LAMA84 were cultured in 10% serum RPMI medium under hypoxic (3% O2) or normoxic (21% O2) conditions. All samples were treated with imatinib 1μM ± prazosin 13 μM (OCT1 reversible inhibitor) for 24 hs.

Results: Cells treated with imatinib and cultured under hypoxic conditions demonstrated decreased apoptosis and increased cell viability compared to normoxic conditions (K562 Annexin + cells 62% vs 94%, p= 0.003, LAMA84 Annexin + cells 61% vs 92%, p=0.0028). The addition of prazosin almost abrogated imatinib efficacy in normoxic environment but did not modify the effect of imatinib under hypoxic conditions.

Conclusions: Our data confirm that OCT1 is the most important imatinib carrier. Exposure of CML cell lines to an hypoxic environment results in reduced sensitivity to imatinib and this effect wasn’t affected by OCT1 inhibition. Search for underlying mechanisms of these findings are in progress.
MDR-1 Gene and BCRP Are Involved in the Resistance to Imatinib in K-562 Cell Line.
Authors: Lardo Mabel , Gargallo Patricia , Bianchini Michele , Lucero Graciela , Koziner Benjamin , Larripa Irene , Lazarowski Alberto
Published: April 2020

Drug resistance to imatinib in chronic myeloid leukemia (CML) has been linked to BCR/ABL gene amplification or mutations in its tyrosine kinase domain of BCR/ABL gene (Phi). However the role of the ABC-transporters in the multidrug resistant phenotype still remains to be clarified.

Objective: to study the functional expression of multidrug resistance gene-1 (MDR-1) and breast cancer resistant protein (BCRP), in imatinib pharmacoresistant K562 Phi+ cells.

Materials and methods: K562 Phi+ cultured cells (ATCC catN°:CCL-2439) were immunophenotyped by flow cytometry, mRNA transcripts of BCR/ABL and MDR-1 were amplified by RT-PCR and P-glycoprotein (P-gp), the product of MDR-1 gene and BCRP, were investigated by inmunocytochemistry, using specific monoclonal antibodies (P-gp: clones C494 and C219; BCRP: clone BXP-21). Total efflux activity of Rhodamine-123 (Rho-123) and its inhibition by Cyclosporin A (CyA) or 4°C, were evaluated using flow cytometry. The apoptotic effect in K-562 cells after 24hs and 72hs of treatment with imatinib (2μmol.L−1) with or without CyA (3ugm.L−1), was evaluated by staining the cells with 1 mL of a mixture of acridine orange (100 mg.mL−1) and ethidium bromide (100 mg.mL−1) in PBS.

Results: The immunophenotype was: HLA-DR-,CD45++,CD11c−/+,CD13+,CD14−,CD15−/+,CD16−,CD33+,CD34−CD117−/+; CD56−, CD61−/+; CD2−, CD3−, CD4−, CD7−, CD8−, CD10+, CD19−, glyA+ . MDR-1 and BCR/ABL transcripts were highly expressed, and P-gp and BCRP immunostaining were also positive. Rho-123 efflux was inhibited by CyA (3ugm.L−1) or 4°C. Increased apoptotic effect (from 7, 21% to 20, 79%) at 24hs and (11% to 53%) at 72hs was observed with combined imatinib+CyA treatment compared with imatinib alone.(p<0.01*)

Conclusions. Our results suggest that P-gp and BCRP might play an active role in the pharmacoresistance to imatinib treatment in K562Phi+ cells. Combined treatment with Imatinib+CyA (or other more specific inhibitors of P-gp and BCRP) could increase the apoptotic effect in BCR/ABL over-expressing refractory cells.

Figure Figure
ASK1 Is Activated by Treatment with Arsenic Trioxide through Accumulation of Reactive Oxygen Species and May Suppress Apoptosis through NF-kappaB in Leukemia Cells.
Authors: Arai Ayako , Yan Weihua , Ichijo Hidenori , Miura Osamu
Published: April 2020

Arsenic trioxide (ATO), a remarkably effective reagent for treatment of relapsed acute promyelocytic leukemia (APL), is reported to induce differentiation of APL cells at low concentrations and apoptosis at high concentrations. Induction of granulocytic differentiation is considered to be a specific effect of ATO on APL cells. By contrast, ATO commonly induces apoptosis of various tumor cells of hematological malignancies, including chronic myeloid leukemia cells expressing the BCR/ABL fusion kinase, as well as those of solid tumors. It was reported that ATO treatment induced intracellular accumulation of reactive oxygen species (ROS) by inhibiting glutathione peroxidase activity. Accumulated ROS induced a decline in cellular mitochondrial membrane potential, followed by cytochrome c release, caspase 3 activation, and apoptosis of cells. On the other hand, it is well known that adequate dose of ROS is indispensable for proliferation and survival of a variety of cells, including hematopoietic cells. Thus, various intracellular signaling pathways are strictly regulated and activated downstream of ROS to promote or suppress apoptosis, and the signaling pathways activated by ROS accumulation induced by ATO need to be defined to understand the mechanisms for ATO-induced apoptosis.

Apoptosis signal-regulating kinase 1 (ASK1) is a ubiquitously expressed mitogen-activated protein kinase kinase kinase that activates the JNK and p38 signaling pathways and is involved in regulation of apoptosis. Here we find that ATO treatment of NB4 and K562 leukemia cells induces activation of ASK1. ATO induced accumulation of ROS, and the ASK1 activation was suppressed by cotreatment with an antioxidant, N-acetyl-l-cysteine. Although the Rho family GTPases Rac and cdc42 were activated by ATO, overexpression of their dominant-negative mutants did not suppress ATO-induced ASK1 activation. ASK1 activation was induced most significantly at low concentrations of ATO, where G2/M arrest but not apoptosis was induced. On the other hand, ASK1 activation induced by ATO was barely detectable at high concentrations, where apoptosis was induced significantly. By contrast, JNK and p38 were activated in dose-dependent manners by ATO. Murine embryonic fibroblasts (MEFs) derived from ASK1-deficient mice were more prone to ATO-induced apoptosis than control MEFs. Moreover, ASK1 was activated by ATO in a more sustained manner in ATO-resistant leukemia cell lines than in sensitive cell lines. Finally, a dominant-negative ASK1 mutant reduced ATO-induced NF-kappaB activation in leukemia cells. Together, the results indicate that ASK1 is activated by ATO through ROS generation independent of activation of Rac and cdc42 and may play a negative role in induction of apoptosis, possibly through activation of NF-kappaB.
Notch Signaling Regulates Hematopoietic Stem Cell Homeostasis in the Fetal Liver through a Non-Cell-Autonomous Mechanism.
Authors: Maillard Ivan , High Frances , Shestova Olga , Dedhia Priya , Epstein Jonathan A. , Pear Warren S.
Published: April 2020

Notch is essential for the generation of definitive hematopoietic stem cells (HSCs) during fetal life, but its effects on subsequent HSC self-renewal are controversial. We have shown recently that canonical Notch signaling is dispensable for the maintenance of stem cell function in adult HSCs (Maillard et al., submitted). We now investigate whether Notch regulates HSC homeostasis during fetal development. For this purpose, we have developed a system to track individual Notch-deprived cells through expression of the GFP-tagged pan-Notch inhibitor DNMAML (Maillard et al., Blood 2004). We have used this system to conditionally inactivate Notch signaling at defined time points during development. Specifically, we crossed mice carrying an ubiquitously expressed Tamoxifen(TAM)-inducible Cre recombinase transgene (CAGG-CreERTm) with mice harbouring a Cre-inducible DNMAML construct (DNMAMLf). We injected TAM i.p. at mid-gestation (E11.5), after generation of fetal HSCs. At E13.5, DNMAML-GFP protein expression was induced in 20–45% of hematopoietic progenitors in the Lin-Sca-1hic-Kithi (LSK) fraction. As compared to control littermates, TAM-induced CAGG-CreERTm x DNMAMLf (TICD) fetuses initially showed increased numbers and decreased quiescence of fetal liver LSK cells: at E13.5, TICD LSK progenitors had increased cell size, expressed elevated levels of Sca-1, and showed an increase in the fraction of cells in S-G2M phases of the cell cycle. Significantly, these abnormalities were observed both within the GFP- and GFP+ TICD LSK fractions, indicating non-cell-autonomous disruption of their homeostasis. At subsequent time points (E15.5), TICD fetuses showed a marked decrease in the number of LSK progenitors, most prominently within the CD150+CD48− subset highly enriched for long-term HSCs (LT-HSC), suggesting HSC exhaustion. Histological examination revealed multiple small areas of hemorrhage in TICD tissues consistent with underlying vascular defects. Fetal death was observed at E17.5–18.5. Altogether, our findings show that continuous Notch signaling is absolutely required for normal development during mid-gestation and suggest that Notch controls a key element of the fetal HSC niche that promotes LT-HSC self-renewal and quiescence. Because inactivation of Notch signaling causes phenotypic changes associated with vascular defects, we speculate that Notch regulates critical vascular elements of the stem cell niche in the fetal liver.
Pyrrolo[2,3-b]Pyridinic Derivatives Induce Cell Growth Inhibition of the Myeloblastic HL-60 Cell Line.
Authors: Decrop Maylis , Espanel Claire , Guillard Jerome , Boissier Elodie , Gallay Nathalie , Binet Christian , Viaud-Massuard Marie-Claude , Herault Olivier
Published: April 2020

Cytokins, N6-subsitued adenine derivatives play an important role in many different processes in plant development (Mok et al, Annu Rev Plant. Physiol Plant Mol Biol2001, 52:89–118), including the cell growth and division control, the vegetable cell differentiation with auxin and the storage of various metabolites as alkaloids (Yahia et al, Plant Science1998, 133:9–15). Furthermore, several studies suggest that cytokinins and their purin derivated are able to control mammalian cell apoptosis and differentiation of human leukemic cells (HL-60 myeloblastic cell line) into mature granulocytes (Ishii et al, Cell Growth Differ2002, 13: 19–26). All these compounds exert their biological activity via Cyclin-Dependant Kinase (CDK) inhibition and particulary CDK1 and CDK2. The aim of our study was the synthesis of new 7-azaindole derivates as cytokinin analogues and the evaluation of their biological effects on HL-60 cells.

Eight analogues of 7-azaindole were prepared by the condension of (N-methyl-)4-chloropyrrolo[2,3-b]pyridine with corresponding amines using palladium-catalyzed reaction (Hartwig et al, Angew Chem IN Ed1998, 37: 2046–2067). The four derivates from 1H-pyrrolo[2,3-b]pyridine were 4-phenylamino-7-azaindole, 4-benzylamino-7-azaindole, 4-phenethylamino-7-azaindole and 4-phenylpiperazylamino-7-azaindole. The four derivates from 1-methyl-pyrrolo[2,3-b]pyridine were 4-phenylamino-N-methyl-7-azaindole, 4-benzylamino-N-methyl-7-azaindole, 4-phenetylamino-N-methyl-7-azaindole and 4-phenylpiperazylamino-N-methyl-7-azaindole. HL-60 cells were exposed to three concentrations of these compounds (10–100–500 μM) during 72h at 37°C. The number of viable cells was determined by Trypan blue exclusion, and the cell cycle was assessed by propidium iodide staining followed by flow cytometric analysis. All these compounds decreased the number of viable cells. The compounds of the NH serie were more active than their methyl-analogues, especially 4-phenylamino-N-methyl-7-azaindole and 4-phenethylamino-7-azaindole which presented an estimated IC50 of 2 μM. Moreover, when used at 10 μM, 4-phenylamino-N-methyl-7-azaindole induced apoptosis whereas 4-phenethylamino-7-azaindole promoted inhibition of the cell cycle without pro-apoptotic effect.

These results suggest that cytokinin analogues derived from 1H-pyrrolo[2,3-b]pyridine may present interesting therapeutic potential as cytostatic agents. Further studies will clarify their biological effects on leukemic cells.
ARRY-429520, a Novel KSP Inhibitor with Potent Anti-Proliferative Activity in Leukemic Cell Lines.
Authors: Lemieux Christine , DeWolf Walt , Voegtli Walt , Wallace Eli , Woessner Rich , Corrette Chris , Allen Shelley , Hans Jeremy , Zhao Qian , Aicher Tom , Lyssikatos Joe , Robinson John , Koch Kevin , Winkler James , Gross Stefan
Published: April 2020

Kinesins are eukaryotic microtubule-associated motor proteins. There are over 40 known kinesins, approximately 15 of which are closely associated with mitosis. Kinesin Spindle Protein (KSP), also known as Eg5, is a mitotic kinesin that is a required enzyme in mitosis (prophase / prometaphase). This protein plays a key role in the formation of the bipolar spindle, particularly related to its role in centrosome maturation/separation. Because KSP is expressed predominately in proliferating cells and is absent from postmitotic neurons, its inhibition should not produce the peripheral neuropathy associated with traditional microtubule disruption agents (taxanes and vinca alkaloids). We report here the in vitro characterization of a potent KSP inhibitor, ARRY-429520, a member of a series of KSP inhibitors discovered and optimized by structure-based design. ARRY-429520 inhibits human KSP, with an IC50 of 6 nM, by a mechanism which was demonstrated to be uncompetitive with respect to ATP and noncompetitive with respect to tubulin. It was shown to arrest cells in mitosis as measured by FACs analysis as well as the accumulation of phospho- histone H3, with an EC50 of 1.5 nM. Furthermore, this compound was demonstrated to be antiproliferative, with EC50s between 0.3 nM and 6.5 nM against a panel of human tumor cell lines, including various leukemia lines (K-562, KU-812, HL-60, KG-1, MOLT3, MOLT4). In addition, ARRY-429520’s potency in MDR-overexpressing cell lines was minimally impacted as compared to paclitaxel. Cellular imaging studies demonstrate that the normal mitotic spindle configuration was disrupted, with the formation of monopolar spindles, a hallmark feature of KSP inhibition, at single digit nanomolar concentrations of ARRY-429520. Markers of mitotic arrest and apoptosis were demonstrated in tumor xenografts from animals treated with ARRY-429520.
Sulforaphane Switches Cyclin E Isoform Expression and Blocks Leukemic Cell Cycling.
Authors: Gorham Laura M. , Frye Gigi , Miranda Michelle , Steinman Richard A.
Published: April 2020

Sulforaphane (SFN), an isothiocyanate derived from cruciferous vegetables, has been shown to inhibit the growth of prostate cancer cells in vitro and in vivo. We were interested in exploring potential antileukemic effects of SFN. The viability of multiple myeloid leukemic cell lines was decreased by 25uM SFN. Pharmacokinetic studies reported in rats suggest that this serum concentration can be achieved through oral dosing. Lower SFN concentrations (1–5 uM) inhibited leukemic cell growth without affecting cell viability. Synchronized HL-60 cells exposed to 25uM SFN were blocked at the G1/S phase transition. Kinetic analysis of cell cycle proteins demonstrated that the G1/S block arose from downmodulation of cyclins D3 and E rather than upregulation of cdk-inhibitors. Interestingly, we found that HL-60 cells expressed a low molecular weight (LMW, 36 kD) variant of cyclin E rather than (50 kD) full-length cyclin E. Treatment with SFN for as little as 2 hours caused a decrease in expression of the LMW cyclin E and induced the expression of a higher molecular weight (~50 kD) cyclin E isoform. Because LMW cyclin E has been associated with increased cdk2 activity and p27 resistance compared to full-length cyclin E, we postulate that SFN-mediated cyclin E isoform-switching contributed to growth inhibition of these leukemic cells. The signaling pathway through which SFN altered cyclin E expression appeared to be distinct from MEK/ERK and JNK pathways that have been implicated in the apoptotic effects of SFN. Given that cyclin E overexpression and, particularly, LMW cyclin E expression are correlated with poor prognosis in multiple cancers, the mechanism through which SFN decreases LMW cyclin E expression in these leukemic cells could have therapeutic significance.
Experimental Study on DNA Molecular Structure of Acute Myeloid Leukemia and Myelodysplastic Syndrome by FT-IR Spectra.
Authors: Wang Shihong , Xiao Zhijian , Han Ming-Zhe , Wu Qiang
Published: April 2020

So for, there were no definite molecular markers in most acute myeloid leukemia(AML) and myelodysplastic syndrome(MDS), and their molecular mechanisms have not been clarified. Their clinical diagnosis were mainly based on morphology of bone marrow and peripheral blood cells. The current study aimed to explore the mechanisms of abnormal DNA molecular structure in AML and MDS by Fourier Transform Infrared(FT-IR) spectra, and to explore the methods of early diagnosis and prognosis. At first, we segregated the mononuclear cells of AML-M2a, MDS and control groups samples, extracted the DNA of these cells, then we checked the completely dry DNA by FT-IR spectra and analysed the identity and difference among AML-M2a, MDS and Control groups. Comparing the mean DNA spectra of the AML-M2a, MDS and control groups revealed statistically significant difference(p<0.001). Each group has an almost concordant FT-IR spectra rule. Compared with the control group, AML-M2a and MDS both were significant different(p<0.001). Absorbance increasing of DNA in AML-M2a, MDS at the wavenumber 2880cm-1 and 2940cm-1 shown hypermethylation of nucleotide base(p<0.001). Absorbance decreasing at wavenumber 1420cm-1 demonstrated deacetylation(p<0.001). Absorbance decreasing at wavenumber 1150cm-1 indicated the abnormality of C—O,S=O,C—OH,C—C,C—O—C,C—N, and the absorbance increasing at wavenumber 1660–1590cm-1 reflected in-plane ring, C=N stretching vibrations and hypermethylation of cytosine. There were also other mild difference among AML-M2a, MDS and control groups. In summary, The principal chain, phosphonic backbone and nucleotide base of DNA in AML-M2a and MDS have significant difference with the control and could be the initial changes of DNA in AML and MDS occurrence and development. Based on the rules of change, FT-IR spectra is a potential method of early diagnosis and prognosis in AML and MDS.