bluuebunny/crossref_metadata_2025 huggingface dataset:
Implication of AML1/RUNX1 Function in the Homeostasis and Leukemic Transformation of Hematopoietic Stem Cells.
Authors: Ichikawa Motoshi , Takeshita Masataka , Goyama Susumu , Asai Takashi , Nitta Eriko , Nakagawa Masahiro , Kawazu Masahito , Chiba Shigeru , Ogawa Seishi , Kurokawa Mineo
Published: October 2019

Transcription factor AML1/RUNX1, initially isolated from the t(8;21) chromosomal translocation in human leukemia, is essential for the development of multilineage hematopoiesis in mouse embryos. AML1 negatively regulates the number of immature hematopoietic cells in adult hematopoiesis, while it is required for megakaryocytic maturation and lymphocytic development. However, it remains yet to be determined how AML1 contributes to homeostasis of hematopoietic stem cells (HSCs). To address this issue, we analyzed in detail HSC function in the absence of AML1. Notably, cells in the Hoechst 33342 side population fraction and c-Kit-positive cells in the G0 cell cycle status were increased in number in AML1-deficient bone marrow, which suggests enrichment of quiescent HSCs. We also found an increase in HSC number within the AML1-deficient bone marrow using limiting dilution bone marrow transplantation assays. Thus, the number of quiescent HSCs is negatively regulated by AML1, loss of which may result in accumulation of leukemic stem cell pool in AML1-related leukemia. To identify mechanisms through which functional loss of AML1 exerts leukemogenic potential, we focused on the AML1-Evi-1 chimeric protein, which is generated by the t(3;21) chromosomal translocation and disturbs the normal function of AML1. We introduced AML1-Evi-1 and its mutants into murine bone marrow cells, and evaluated hematopoietic cell transformation by colony replating assays. The transforming activity of AML1-Evi-1 was impaired when any of the major functional domains of AML1-Evi-1 was lost. Moreover, overexpression of Evi-1 could not transform AML1-deleted bone marrow cells, suggesting that fusion of AML1 and Evi-1, rather than AML1 suppression and Evi-1 overexpression, is essential for AML1-Evi-1 leukemogenesis. Intriguingly, among the hematopoietic progenitor cell fractions, AML1-Evi-1 could transform only the uncommitted, immature hematopoietic cells, which contrasts with MLL-ENL, a chimeric protein generated in t(11;19) leukemia. AML1-Evi-1 transformed cells show a surface marker profile different from that of the cells transformed by AML1-MTG8/ETO, another leukemic gene product that also perturbs AML1 function. These results provide a valuable clue to a distinct mechanism determined by the Evi-1 moiety in the AML1-Evi-1 leukemogenesis and to a role of AML1 loss in the self-renewal of leukemic stem cells.
GATA and BCLxl Downregulation in Erythropoiesis during In Vitro Lineage Specific Differentiation of MDS Hematopoietic Progenitor Cells Is Not Induced by Activated Notch Pathway.
Authors: Hopfer Olaf J. , Komor Martina , Koehler Ina S.N. , Freitag Claudia , Hoelzer Dieter , Thiel Eckhard , Hofmann Wolf-Karsten
Published: October 2019

Notch signals have recently been shown to inhibit erythroid and megakaryocytic differentiation of hematopoietic progenitor cells. In myelodysplastic syndrome (MDS) its role in dyserythropoiesis has not been fully elucidated. Therefore we asked whether dysregulation of Notch pathway elements might be associated with impaired GATA1 and BCLxl expression and ineffective erythropoiesis being a hallmark of MDS hematopoiesis. We have generated an in-vitro model of MDS lineage-specific hematopoietic differentiation by culturing CD34+ bone marrow cells from healthy donors (n=7) and MDS patients (low risk: RA/n=6, RARS/n=3; high risk: RAEB/n=4, RAEB-T/n=2) with EPO and TPO. Cell harvest was at days 0, 4, 7 and 11. Expression of GATA1, BCLxl, DLK1, Notch1, HES1 and HERP2 was measured by real time RT-PCR (qPCR). RNA expression of GATA1 and of BCLxl was steadily upregulated, particularly during late normal erythropoiesis. During normal megakaryopoiesis expression of both genes was up to 50 times lower as compared to normal erythropoiesis. In contrast, during MDS erythropoiesis a loss of typical late upregulation of GATA1 and BCLxl was observed. DLK1 expression during erythropoiesis showed increased expression particularly in high risk MDS vs. normal controls. Expression of HES1 was increasing during the course of normal erythropoietic and megakaryopoietic differentiation but not in lineage specific cells from MDS patients. In conclusion our data show that the central erythropoietic transcription factor GATA1 and the associated antiapoptotic molecule BCLxl are markedly downregulated during MDS erythropoiesis which may contribute to the ineffective erythropoiesis seen in this disease. Increased DLK1 expression in differentiated stem cells from high risk MDS patients was seen. However, an upregulation of the Notch pathway leading to increased expression of the GATA1 repressor HES1 could not be detected.
The Expression of MT1-MMP in Human Hematopoietic Cell Lines and Its Influences on the Biological Behaviors of HL60.
Authors: Jiang Juxiang , Ma Zhenni , Dong Ningzheng , Bai Xia , Ruan Changgeng
Published: October 2019

Membrane type matrix metalloproteinase-1(MT1-MMP) is the identified member of characterized MMPs. Recent researches suggest that MT1-MMP participates in the progress of hematology malignancies, but the underlying mechanisms have not been clearly defined. To explore the effects of MT1-MMP in hematology malignancies, the expression of MT1-MMP as well as MMP-2 and TIMP-2 in 13 malignant hematopoietic cell lines was investigated and its eukaryotic expression vector was constructed and tranfected to HL60 with low expression of MT1-MMP. mRNA expressions of MT1-MMPs, MMP-2 and TIMP-2 in hematopoietic cell lines with different lineages were semi-quantified by RT-PCR. The gelatinolytic activity of MMP-2 was visualized on gelatin zymograms and the expression of MT1-MMP protein was detected by flow cytometry. The eukaryotic expression vector pcDNA3.1/V5-His-TOPO-MT1-MMP was constructed and identified by enzyme digestion, PCR and sequencing, the vector was transfected into HL60 with liposomes and the transfection efficiency was analyzed by RT-PCR, real-time PCR and flow cytometry. HL60/pcDNA3.1 cells were adopted as controls. Cell proliferation was examined by MMT, and expressions of MMP-2, Bcl-2 and Bax were analyzed by real-time PCR. Invasive potential and cell cycle of the transfected cells were studied with transwell plate and flow cytometry. The expression patterns of the MT1-MMP/MMP-2/TIMP-2 were varied in 13 hematopoietic cell lines. No obvious correlation between the origin of the cells and the expression level of the detected genes was observed (P>0.1), while MT1-MMP significantly correlated to MMP-2 (R=0.64, P=0.02). The expression vector of MT1-MMP was successfully constructed. Significantly enhanced proliferative ability was observed in HL60/MT1-MMP than in control (P<0.01). No obvious difference was observed as to the expression of MMP-2, Bcl-2 and Bax between the two groups. At 48h after transfection, significantly increased invasiveness was noted in HL60/MT1-MMP (15.42±2.38%)than in control(5.16±0.78%)(P<0.01). At 24h after transfection, higher ratio of G2 and S phase cells was found in the experiment group than in control (67.5±3.57% vs 57.9±2.52%) (P<0.05), whereas at 48h and 72h after transfection, no obvious difference of cell cycle was observed between the two groups. The association between expression of MT1-MMP and MMP-2 in malignant hematopoietic cell lines may suggest the close relation of their functions. Over-expressing MT1-MMP in leukemic cell lines exhibit enhanced proliferative and invasive potential. No direct relationship can be identified between MMP-2 and enhanced invasiveness. Directive involvement of MT1-MMP may be absent in the regulation of apoptosis induced by the Bcl-2/Bax pathway.
Two Novel Translocations That Disrupt the RUNX1 Gene in Acute Myeloid Leukemia.
Authors: Dai Haiping , Xue Yongquan , Pan Jinlan , Wu Yafang , Wang Yong , Shen Juan , Zhang Jun
Published: October 2019

Translocations involving 21q22 are commonly observed in both de novo and therapy-related acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS). They often result in the disruption of RUNX1 and give rise to fusion genes consisting RUNX1 and different partner genes which contribute to leukemogenesis. At present, at least 21 such translocations have been reported in the literature. We report here two novel translocations involving the RUNX1 gene: t(1;21)(q12;q22) in a 53-year-old female with AML-M5b and t(11;21)(q13;q22) in a 65-year-old male with AML-M2. The abnormalities revealed by R-banding karyotypic analysis were confirmed by interphase and metaphase fluorescence in situ hybridization (FISH), chromosome painting (CP), and M-FISH.
Hidden Abnormalities and Novel Classification of t(15;17) APL Based on Genomic Alterations.
Authors: Akagi Tadayuki , Ogawa Seishi , Yamamoto Go , Sanada Masashi , Kawamata Norihiko , Miller Carl W. , Liang Der-Cherng , Shih Lee-Yung , Koeffler H. Phillip
Published: October 2019

Acute promyelocytic leukemia (APL) is a hematopoietic malignant disease characterized by the chromosomal translocation t(15;17), resulting in the fusion of PML-RARA genes. We examined whether genomic alterations could be found other than t(15;17) using a sensitive technique in order to subcategorized this disease on the basis of genomic status. Thirty-three t(15;17) APL samples were analyzed with high-density single-nucleotide polymorphism microarray (50K SNP-chip) using the new algorithm AsCNAR (allele-specific copy-number analysis using anonymous references). Our analysis revealed that 10q (2 cases), 11p (3 cases) and 19q (1 case) regions were identified as loss of heterozygosity with normal copy number [we call this somatic uniparental disomy (UPD)] that could not be detected by karyotypic analysis. Nineteen samples (58%, Group A) did not have an obvious alteration. Fourteen samples (42%) showed either one or more genomic abnormalities: 6/14 of these samples (18%, Group B) had trisomy 8 either with or without duplication, deletion, and UPD; and 8/14 of these samples (24%, Group C) had abnormalities without trisomy 8. Interestingly, FLT3-ITD mutation (7/33 cases) was found only in Group A. These results suggest that the pathway of development of APL differs in each group; FLT3-ITD, trisomy 8 (probably c-Myc gene), and unknown factor(s) are involved in group A, B, and C, respectively. Here, we showed for the first time hidden abnormalities and novel disease-related genomic regions in t(15;17) APL. Our technique may become a routine, rapid, robust technique for subclassification of APL and to screen for novel therapeutic targets.
Bio-Cell Chip Method for Stable DNA Storage and Simple Extraction.
Authors: Kim Miyoung , Lee Dong Soon , Kim Tae Young , Oh Bora , Min Hyun Jung , Yoon Sung-soo
Published: October 2019

We have previously developed the bio-cell chip, on which cells are arrayed and immobilized on a glass slide, using multi-well device. Development of bio-cell chip was originally targeted for the application in fluorescent in situ hybridization and immunohistochemistry. Here, we suggest simple and convenient method of DNA extraction from bio-cell chip, which can handle hundreds of specimens. One uL of cell suspension was loaded on wells of the bio-cell chip and after drying, 2uL of BR-A buffer (GenScript BloodReady™Multiplex PCR System) was dropped onto the wells of bio-cell chip. After incubation for 10 minutes, each mixture was transferred to a microtube with additional buffer to a total volume of 20uL. One uL of mixture was taken and 20uL of mixture for PCR reaction was prepared; 1U BioTherm™DNA Polymerase (Genecraft, GERMANY), 1uL (10pM/ul) of each oligonucleotide primer for target sequence, 2.5mM dNTP, and 1.0uL of DNA mixture. PCR for GST M1 and hGAPDH was carried out using the MJ Research DNA thermal cycler. With this bio-cell chip extraction method, simplification of experimental procedure not only lessened the loss of specimen but also saved the time. Only 10 min of reaction time and a very small volume of samples was required without any further purification step. Amplification of DNA was possible, reflecting safety in DNA stability. Requirement for the diagnostic specimen transport is intricate; strict regulations on infectious materials and preparation of specimen package, cell fixatives such as methanol. Bio-cell chip enables transport of hundreds of samples at a thin plate without transportation media, and also simple extraction of DNA from bio-cell chip with high throughput efficiency, with excellent stability comparable to those with fresh samples.

Figure Figure
A Novel Method of Microarray Quantitave Analysis of P16 CPG Island Methylation in Leukemia Cells from Patients with Hematological Malignancies.
Authors: Shou Beiming , Chen Baoan , Zhou Dongrui , Ding Jiahua , Gao Chong , Sun Yunyu , Wang Jun , Chen Jian , Zhao Gang , Ma Yan , Song Huihui , Bao Wen , Pei Xiaoping , MA Xudong , Liu Delong
Published: October 2019

p16 is a tumor suppressor gene frequently affected by hypermethylation of promoter CpG island in malignant disorders. Phenylhexyl isothiocyanate (PHI) has been shown to induce p16 hypomethylation in leukemia cells. However, the methylation status is usually studied qualitatively or semi-quantitatively at the best. In this study, we developed a novel method to quantify p16 methylation in clinical specimen from patients with hematological malignancies as well as in U266, a multiple myeloma cell line. To establish a standard curve of p16 promoter methylation, methylated and demethylated PCR products were subcloned into plasmids respectively. The clones were selected out as completely methylated and demethylated clones, respectively. The PCR products of the two clones were mixed at different ratios and analyzed on a mciroarray chip to produce a standard curve of methylation. p16 promoter methylation was determined in U266 cells. The baseline p16 methylation was 78.2% in U266 cells. The methylation was decreased to 61.7% and 54.8%, respectively in the presence of PHI of 5 and 10 uM, respectively. We further analyzed p16 methylation status in 7 patients’ specimen, two from blood, 5 from bone marrow. Two patients had ALL, two had AML, one CLL, one MM, and one CMML. 3 patients have p16 gene hypermethylation at 17.7%, 47.5% and 84.3%, respectively. Hypomethylation occurred in two of the three patients’ cells after PHI treatment in vitro (84.3% to 64.8%, and 47.5% to 25.4%). In conclusion, a quantitative microarray method was established for quantitation of p16 methylation status. This will be used for laboratory screening of hypomethylation drugs, such as PHI, and for clinical application to monitor therapeutic efficacy of hypomethylating drugs, such as azacitidine, and to prognostify patients based on p16 methylation level.
RUNX1/EVI1 That Blocks Myeloid Differentiation Inhibits C/EBPα Function.
Authors: Tokita Katsuya , Maki Kazuhiro , Mitani Kinuko
Published: October 2019

RUNX1/EVI1 chimeric transcription factor produced by t(3;21) causes leukemic transformation in hematopoietic stem cell tumors such as chronic myelogenous leukemia (CML) in blastic crisis and myelodysplastic syndrome (MDS) in leukemic transformation, possibly through differentiation block of malignant myeloid progenitors. We have recently reported that Runx1/EVI1 knock-in heterozygous mice show defective hematopoiesis in the fetal liver similar to Runx1 knock-out mice, but possess dysplastic hematopoietic progenitors with high self-renewal capacity. Notably, Runx1/EVI1 knock-in chimeric mice developed acute megakaryoblastic leukemia. The molecular characterization of RUNX1/EVI1 points two major functions; one is dominant-suppressive function over wild-type RUNX1 and the other is EVI1’s own function; blockade of TGFb-mediated signal, inhibition of JNK and stimulation of AP-1 activity. C/EBPa is a key transcriptional regulator that induces the granulocytic differentiation of myeloid progenitors and several lines of evidence suggest that disturbance in C/EBPa signaling is one of the major molecular events in myeloid malignancies. In this study, we investigated whether RUNX1/EVI1 affects the expression and function of C/EBPa. We introduced RUNX1/EVI1 cDNA into LG-3 cells that differentiate along the myeloid lineage upon granulocyte colony-stimulating factor exposure, and confirmed that RUNX1/EVI1 suppressed the differentiation. To further investigate the molecular mechanisms of RUNX1/EVI1-mediated differentiation block, we analyzed RUNX1/EVI1’s effect on the functions of C/EBPa. RUNX1/EVI1 was found to associate with C/EBPa. By using the reporter assay with the CEBPA promoter, we observed a dominant-negative effect of RUNX1/EVI1 over C/EBPa-mediated transcriptional activation via the CtBP-binding site in the EVI1 portion. In the gel-shift assay, RUNX1/EVI1 down-regulated DNA-binding activity of C/EBPa. Therefore, recruitment of histone deacetylase via CtBP and disruption of DNA binding could be likely scenarios for the RUNX1/EVI1-induced dominant repression on C/EBPa. Importantly, co-expression of C/EBPa restored differentiation ability of the RUNX1/EVI1-expressing LG-3 cells. All these data argue that inhibition of C/EBPa function may be causatively related to the RUNX1/EVI1’s leukemogenic potential.
Defective Activation of Phospholipase Cγ2 by Collagen in Platelets Lacking the Semaphorin Family Member, Sema4D.
Authors: Zhu Li , Jiang Hong , Kumanogoh Atsushi , Kikutani Hitoshi , Brass Lawrence F.
Published: October 2019

Semaphorins are a large family of cell surface molecules best known for their ability to mediate communication between cells during neural development. We have recently shown that human platelets express the semaphorin family member, sema4D, and both of its known receptors, CD72 and plexin-B1 (Zhu, et al, PNAS, 2007). We have also shown that sema4D(−/−) mice have an impaired response to arterial injury in vivo and a selective defect in collagen- and convulxin-induced platelet aggregation in vitro. In the present studies we have sought the molecular basis for these defects, focusing on events downstream of glycoprotein VI (GPVI), which serves as a receptor for both collagen and convulxin. In normal platelets, GPVI signaling leads to the phosphorylation and activation of phospholipase Cγ2 (PLCγ2) through the formation of a signaling complex that includes SLP-76 and LAT. This complex is activated when GPVI-associated FcRγ is phosphorylated, allowing the tyrosine kinase, Syk, to bind. PLCγ2 activation results in phosphoinositide hydrolysis, an IP3-mediated increase in cytosolic Ca++, and activation of additional kinases, such as Akt. In theory, the absence of sema4D could affect any of these steps and by doing so impair collagen-induced platelet aggregation. Working backwards through the GPVI pathway, our results showed that compared to platelets from matched WT mice, sema4D(−/−) platelets have 1) a rightward-shift in the dose/response curve for collagen-induced Akt phosphorylation, 2) a 37% smaller increase in cytosolic Ca++, and 3) a 43% smaller increase in PLCγ2 phosphorylation. However, we found no defect in collagen-induced FcRγ phosphorylation, which is the earliest event in GPVI signaling. The defect in PLCγ2 phosphorylation was not limited to mouse platelets, but was also observed when human platelets were stimulated with collagen in the presence of an antibody directed towards the sema4D extracellular domain. Taken together, these results show that sema4D is needed for optimal activation of PLCγ2 by collagen downstream of the GPVI/FcRγ complex. Sema4D is believed to act in part through contact-dependent binding of sema4D to its receptors, CD72 and plexin-B1. Since these studies were performed under conditions in which platelet:platelet contacts can occur, the observed defect in collagen and convulxin responses could be due to impaired signaling by either of these receptors or, in theory, by retrograde signaling via sema4D. One candidate mechanism involves a regulatory complex between CD72 and the tyrosine phosphatase, SHP-1, which we have shown to occur in resting human platelets and to be lost when platelets are activated by agonists or stimulated by soluble sema4D. In theory, sema4D-dependent loss of the CD72/SHP-1 complex allows SHP-1 to relax into an inactive conformation, promoting protein tyrosine phosphorylation, which would not occur when sema4D is absent or blocked.
Stat5a Is Epigenetically Silenced by Oncogenic Tyrosine Kinase NPM/ALK and Acts as Tumor Suppressor by Reciprocally Inhibiting Expression of NPM/ALK.
Authors: Zhang Qian , Wang Hong Yi , Liu XiaoBin , Wasik Mariusz A.
Published: October 2019

Although Stat5a and Stat5b exhibit some non-redundant functional properties, their distinct contributions to carcinogenesis are not clearly defined. Here we report that Stat5a expression is selectively inhibited by DNA methylation of the Stat5a gene promoter region in cells expressing an oncogenic tyrosine kinase NPM/ALK. The DNA methylation is induced by NPM/ALK itself by employing its key effector Stat3, and is associated with binding to the promoter of MeCP2 capping protein and lack of binding of the Stat5a transcription activator Sp-1. Reversal of the methylation by DNA methyltransferase inhibitor, 5′-aza-2-deoxycytidine, restores the Sp-1 binding and Stat5a expression. Strikingly, the induced or exogenously expressed Stat5a binds to enhancer and exon 14 of the NPM/ALK gene and triggers selective suppression of NPM/ALK expression. These results demonstrate that NPM/ALK induces epigenetic silencing of Stat5a and that Stat5a can act as key tumor suppressor by reciprocally inhibiting expression of NPM/ALK.
A Study of Multiple Drug Resistance Mechanisms Improved Against Bortezomib on Multiple Myeloma Cell Lines In Vitro.
Authors: Uyuklu Tolga , Ural A. Ugur , Sarper Meral , Avcu Ferit , Baran Yusuf , Elci Pinar , Akar Nejat
Published: October 2019

The most important problem in the treatment of Multiple Myeloma (MM) is the multi drug resistance (MDR) observed before and after the treatment. For this reason in MM cases an early resistance to treatment can be developed or the disease can relapsed in early period. Yet, there has been no improved drug resistance against proteazom inhibitor Bortezomib (Bor), which is used alone or with other chemotherapeutic agents in resistant or relapsed MM cases. In this study, bortezomib resistant human MM cell lines; RPMI-8226, secreting lambda light chain, and ARH-77, secreting IgG, were developed and responsible resistance mechanisms were investigated. For this purpose, by exposing to the cells to sequentially gradual doses of Bor in vitro conditions, resistant cell lines were acquired throughout one year. The IC50 values for Bor were determined after 48 hour incubation by MTT cytotoxicity assay (IC50:1,16nM for RPMI-8226 and IC80:0,6nM for ARH-77) against wild type cells. Throughout one year some cell lines resistant to 1,3nM Bor were acquired by performing Bor to both cell lines in gradual doses. In resistant cell lines IC50:18,07 for RPMI-8226 and IC50:97,56 nM for ARH-77 were determined by MTT assay. In parallel of the gradual increase in drug concentration; the expression changes of the genes of ATP binding cassette protein; MDR1 (Multi Drug Resistance Protein), MRP1 (Multi Drug Resistance Associated Protein), BCRP (Breast Cancer Resistance Protein); and LRP (Lung Resistant Protein) which is responsible for accumulation of the drug in cytoplasm with the aid of nuclear membrane were determined with Reverse Transcriptase Polymerase Chain Reaction (RT-PCR) and densitometric analysis. In resistant cells, high expression of MDR1, MRP1, BRCP and LRP genes showed that; pumping the drug out of the cell membrane and decrease in accumulation of the drug in the cytoplasm had effects on the resistant mechanisms against Bor. Furthermore, expression changes of an important sing of apoptosis ‘caspase-3’, pro-apoptotic ‘bax’ and an anti-apoptotic ‘bcl-2’ genes were examined by RT-PCR and we could come to a point that when compared the sensitive cells to resistant cells, expression of caspase-3 gene and pro-apoptotic bax protein decreased but bcl-2 gene expression increased in resistant cell lines. Finally, we concluded that resistant cell lines acquired resistance against apoptosis by means of mitochondria. By means of this project, the genes which are responsible for secondary drug resistance in ARH-77 and RPMI-8226 MM cell lines in vitro conditions against Bor were determined. Also resistance mechanisms against apoptosis were demonstrated. Cross resistance to different chemotherapeutic agents mechanisms are still continuing.
Identification of LIV1, a Zinc Transporter Gene, Responsible for HDACI-Induced Apoptosis, Using a Genetic Approach.
Authors: Zhou Jianfeng , Wang Beibei , Chen Pingbo , Wang Jintao , Zhang Yicheng , Ma Ding , Ma Xiaoli
Published: October 2019

Histone deacetylase inhibitors (HDACI) show promise as chemotherapeutic agents and have been demonstrated to induce apoptosis of tumor cells. To gain a better understanding of the molecular basis for HDACI-induced apoptosis and directly identify the gene products which are essential for mediating the apoptosis-regulatory functions of HDACI, we employed the suppression of mortality by antisense rescue technique (SMART). This approach permits the isolation of apoptosis-associated genes based on their selective inactivation by overexpression of antisense cDNAs. Because the antisense mRNA inactivates gene expression of apoptosis-specific genes, transfected cells survive in the presence of apoptosis inducers. We isolated several genes associated with HDACI-induced mortality (GHIM). Here we characterized one of the GHIM cDNAs, GHIM-27. Sequence analysis suggests that the GHIM-27 product is identical to human zinc transporter LIV1. Further researches indicate that LIV1 is significantly induced by HDACI treatment in various cancer cell lines. Down-regulation of LIV1 confers a strong resistance against HDACI-induced apoptosis, which is caused at least in part by disregulation of intracellular zinc homeostasis. Together, these studies identify a zinc transporter gene LIV1 which is responsible for HDACI-induced apoptosis by using a genetic approach. This may provide a potentially important target for anticancer therapy.
Exogenous Nucleic Acids - A Potential Source of Resistance to Nucleoside Analogues in Cancer and Antiviral Therapy.
Authors: Buhmann Raymund , Yang Ting , Obermeier Martin , Jaeger Gundula , Kolb Hans-Jochem
Published: October 2019

The metabolism of exogenous nucleic acids is not well defined. Moreover, there is little information whether there might be interference with chemically and structurally related drugs, e.g. nucleoside analogues (NA) widely used in cancer or in antiviral therapy. In the present report we provide evidence, that nucleic-acid based drugs might antagonize fludarabine or acyclovir. In vitro, fludarabine treated lymphocytes or myeloid blasts where rescued from apoptosis when incubated with defibrotide (DF), a polydisperse mixture of single-stranded oligodeoxyribonucleotides (15 to 30 kD) or singular deoxynucleotides. Thereby deoxycytidine (dCTP) turned out to be the key substrate competing with fludarabine for phosphorylation by deoxycitidine kinase (dCK) and suggested interference with nucleic acid metabolism rather than direct competition with the drug. To further prove this hypothesis the influence of defibrotide on HSV replication was evaluated. In standard drug resistance assays performed with acyclovir sensitive herpes simplex virus (HSV) strains (V0631508) 4 mM of DF restored viral replication in presence of 50 mM acyclovir. This was confirmed by quantitative PCR of viral DNA. Moreover, in this case deoxythymidine (dTTP) turned out to be the competitor for intracellular phosphorylation mediated by virus thymidine kinase. We conclude that treatment with DF and other nucleic-acid-based drugs interfere with the efficacy of NA used for cancer and antiviral therapy. Prospective clinical trials are required to confirm these in vitro findings.
Mutations of the Genes for Ribosomal Proteins L5 and L11 Are a Common Cause of Diamond-Blackfan Anemia.
Authors: Gazda Hanna T. , Sheen Mee Rie , Darras Natasha , Shneider Hal , Sieff Colin A. , Ball Sarah E. , Niewiadomska Edyta , Newburger Peter E. , Atsidaftos Eva , Vlachos Adrianna , Lipton Jeffrey M. , Beggs Alan H.
Published: October 2019

Diamond-Blackfan anemia (DBA), a form of congenital red cell aplasia with marked clinical heterogeneity and increased risk of malignancy, has been associated with mutations in ribosomal protein (RP) gene RPS19 in 25% of probands and in RPS24 or RPS17 in ∼2% of patients. Thus, DBA appears to be a disorder of ribosome synthesis. To test the hypothesis that mutations in other RP genes may also cause DBA, we carried out direct sequencing of candidate RP genes. Genomic DNA samples from 96 unrelated DBA probands (14 familial and 82 sporadic cases) without RPS19 or RPS24 mutations were screened for mutations in RPS3a, RPS13, and RPS16 (previous studies revealed that RPs S19, S24, S3a, S13, and S16 are involved in binding of eIF-2 to the 40S subunit); RP genes L18, L13A, L36, L28, L18A, L40, S5, S9, S11, and S28 (located on chromosome 19); and RP genes, L5, L11, L22, S8, and S27 (on chromosome 1). PCR primers were designed to amplify the coding exons and intron/exon boundaries. We found multiple mutations in two RP genes, L5 and L11. Subsequently we sequenced these two genes in 42 additional DNA samples from DBA probands. In total, we screened 5′UTR, promoter and coding regions, and exon/intron boundaries of RPL5 and RPL11 in 138 DBA unrelated probands. We identified 14 mutations in RPL5 in 138 probands (∼10%), 13 of which are nonsense mutations, deletions or insertions of 1–5 nucleotides causing frameshift and premature termination. One missense mutation, 418G>A, results in a G140S substitution. We found nine mutations in RPL11 in138 DBA probands (6.5%), including five acceptor or donor splice site mutations (introns 1–4) and four deletions or insertions of 1–4 nucleotides causing frameshifts (codons 32-120). None of these sequence changes were found on the NCBI (http://www.ncbi.nlm.nih.gov/SNP/) or the HapMap (http://www.hapmap.org/) SNP lists. Both genes, as well as RPL23 have recently been demonstrated by others to activate the p53 tumor suppressor protein by inhibiting MDM2-mediated p53 ubiquitination and degradation. Moreover, knockdown of any of these genes by siRNAs markedly reduced p53 induction by the ribosomal biogenesis stressor, actinomycin-D. These findings suggest that DBA patients with mutated L5 and L11 proteins may have inadequate p53 pathway activation and (consistent with clinical observations) be at increased risk for neoplasia. We are currently investigating the role of RPL5 and RPL11 mutations in ribosomal biogenesis and in the p53-mediated cell cycle arrest and apoptosis in DBA patients.
Novel Heritable Mutation of the Transcription Factor RUNX1 as a Cause of Autosomal Dominant Familial Platelet Disorder with Predisposition to Acute Myeloid Leukemia (FPD/AML).
Authors: Patton William N. , Suthers Graeme , Altree Meryl , Carmichael Catherine , Wilkins Ella , Carroll Jacqueline , Rawlings Lesley , Scott Hamish
Published: October 2019

Aim To identify the causative heritable mutation in a family with autosomal dominant familial platelet disorder with predisposition to acute myeloid leukemia (FPD/AML).

Method Confirmation of family pedigree, enrolment into ethics committee approved Australian Familial Hematological Cancer Study, procurement of genomic DNA from pedigree members and genetic analysis by sequencing of RUNX1 and CEBPA genes.

Results The proband intially presented aged 50 with mild thrombocytopenia initially diagnosed as idiopathic thrombocytopenic purpura when bone marrow examination (including cytogenetics analysis) was normal. Three years later she was referred with severe progressive thrombocytopenia unresponsive to high dose steroids and intravenous immunoglobulin together with mild anemia and neutropenia. Marrow examination revealed subtle dysplasia with monosomy 7 in 12/20 metaphases. A diagnosis of myelodysplastic syndrome (MDS) was made. Three months later (Feb 2007), she progressed to acute myeloid leukemia (AML). The proband’s mother had had mild thrombocytopenia with subsequent MDS (aged 70) and died of AML two years later. The proband’s only sibling and nephew have mild thrombocytopenia without features of MDS. The proband entered cytogenetic remission following one course of AML induction therapy but continued to show dysplastic features. She then received 2 cycles of consolidation therapy and has undergone unrelated donor allogeneic stem cell transplant (July 2007). Sequencing of PCR products from exons of the RUNX1 gene identified a novel heterozygous mutation in the proband’s constitutional DNA: c.958C>T in exon 7, in the transactivation domain, causing a nonsense mutation, p.Arg293X (sequence variation for RUNX1 classified according to GenBank Accession No. NC_000021). The sibling has the same mutation and studies in other relatives are underway.

Conclusion This study has identified a novel RUNX1 mutation responsible for FPD/AML in this family. Clinicians should be aware of this rare inherited disorder and the availability of genetic testing. People with mutations may be asymptomatic and genetic testing in such families is essential before considering bone marrow transplantation from a living related donor.
Translocation (X;20)(q13;q13.3): Report of Three Additional Cases.
Authors: Sreekantaiah Chandrika
Published: October 2019

We report a recurrent translocation (X;20)(q13;q13.3) in three patients. The translocation was the sole chromosomal abnormality in all three patients and the number of cells with the abnormality varied from three to seventeen out of twenty metaphases analyzed for each patient. The patients were all female with ages ranging from 66 to 83. The presenting symptoms were variable but all included a history of anemia. Bone marrow aspiration showed acute monocytic leukemia in one patient and normocellular bone marrow with no detectable morphologic or immunophenotypic evidence of neoplasm in the other two. Only eight cases with the translocation have previously been reported. Seven of these cases had either myelodysplastic syndrome or acute myeloid leukemia and one patient had pancytopenia of unknown etiology. Repeated bone marrow evaluations on this patient showed no dyspoietic changes. The t(X;20) has clearly been established as a nonrandom abnormality, however, the clinical significance of the translocation is not clear. Close follow up of these patients is therefore essential. Characterization at the molecular level will also help to determine the genes involved and the mechanism of their action.
FBW7 E3-Ligase Mutations Are Associated with T Cell Leukemia.
Authors: Buonamici Silvia , Thompson Benjamin J. , Sulis Maria L. , Palomero Teresa , Ferrando Adolfo , Aifantis Iannis
Published: October 2019

Recent studies have shown that activating mutations of NOTCH1 are responsible for the majority of T cell acute lymphoblastic leukemia (T-ALL) cases. Most of these mutations truncate its C-terminal domain, a region that is important for the NOTCH1 proteasome-mediated degradation. We report that the E3 ligase FBW7 targets NOTCH1 for ubiquitination and degradation. Our studies map in detail the amino acid degron sequence required for NOTCH1-FBW7 interaction. Furthermore, we identify inactivating FBW7 mutations in a large fraction of human T-ALL lines and primary leukemias. These mutations abrogate the binding of FBW7 not only to NOTCH1 but also to the two other characterized targets, c-Myc and cyclin E. The majority of the FBW7 mutations were present during relapse, and they were associated with NOTCH1 HD mutations. Interestingly, most of the T-ALL lines harboring FBW7 mutations were resistant to γ-secretase inhibitor treatment and this resistance appeared to be related to the stabilization of the c-Myc protein. Our data suggest that FBW7 is a novel tumor suppressor in T cell leukemia, and implicate the loss of FBW7 function as a potential mechanism of drug resistance in T-ALL.
Treatment with Valproic Acid Restores Differentiation in AML1/ETO-Positive Leukaemic Cells.
Authors: Zapotocky Michal , Starkova Julia , Mejstrikova Ester , Smetana Karel , Trka Jan
Published: October 2019

In t (8, 21) acute myeloid leukaemia (AML), the leukaemogenesis is supposed to be promoted by interference with expression of AML1 target genes. Repressor complex associated with AML1/ETO fusion protein recruits class I histone deacetylases (HDAC). Valproic acid (VPA) was found to have an extensive effect on AML blasts, via inhibition of class I HDAC. It was shown previously that VPA treatment disrupts the AML1/ETO-HDAC1 complex from AML1 promoter thus leading to apoptosis at different cell lines. However, there is still lack of in-depth morphological and immunophenotypical proof of the hypothesized restoration of differentiation after treatment with VPA. We aimed to characterize the differentiation effect of VPA on AML1/ETO-positive leukaemic cells. Kasumi-1 (AML1/ETO-positive) cell line and MV4-11 (MLL/AF4-positive) cells were treated with VPA and Trichostatin-A (TSA) another HDAC inhibitor. The effect of VPA on cell cycle and differentiation was examined by flowcytometry. DNA, RNA and AgNORs staining of nucleus and nucleolus were used for morphological evaluation. VPA treatment of Kasumi-1 cells resulted in decreased expression of early myeloid progenitor antigens [CD33 (p<0.001), CD34 (p=0.003), CD117 (p<0.001)] and increased expression of antigens typical for more differentiated myeloid cells [CD11a (p=0.001), CD11b (p=0.007)]. Morphological evaluation revealed nucleus fragmentation without chromatin condensation, which evoked monocytoid look of the nucleus in VPA-treated Kasumi cells. The nucleolar morphology and cytochemistry indicated that these cells entered the ageing process and decreased proliferation activity. Large nucleoli characterised by a reduced number of silver-binding nucleolar organizer regions (AgNORs) as well as by their translocation to the nucleolar periphery represented a base for such interpretation. VPA-treated Kasumi cells exhibited accumulation in G1/G0 phase and decreased proliferation (VPA 0.5mM - G1/G0 70%, S/G2/M 16%; control cells - G1/G0 62%, S/G2/M 28%). Conversely, MV4-11 cell line (representing myeloid leukaemic cells of non-CBF origin) did not show any statistically significant differences in imunophenotype after VPA treatment (CD15, CD11b, CD117, CD45, HLA DR, CD14). VPA treated MV4-11 cells did not show accumulation in G1/G0 phase (VPA 0.5mM G1/G0 63%, S/G2/M 13%; control G1/G0 70%, S/G2/M 22%). We show that treatment with VPA restores differentiation of AML1/ETO positive cells as presented by both changes in immunophenotype and morphological analysis. This observed differentiation correlates with decrease in cell cycle. We demonstrate that the effect on differentiation is specific for AML1/ETO-positive cells as it is not noticeable in leukaemic cells of non-CBF origin.
Identification of Differentially Expressed Proteins from the Mitochondria Enriched Cytoplasmic Fractions in Primary AML Cells.
Authors: Shin Myung-Geun , Kim Hye-Ran , Kim Hyeoung-Joon , Lee Il-Kwon , Kim Mi-Ji , Shin Jong-Hee , Suh Soon-Pal , Ryang Dong-Wook
Published: October 2019

Mitochondrial dysfunction has been associated with chronic degenerative diseases, aging and cancer. Mitochondrial proteins are interesting targets for the study of apoptosis in leukemia and other malignancies. Modern mass-spectrometry instrumentation has achieved excellent performance in terms of sensitivity, resolution and mass accuracy; however, so far, the contribution of proteomics to the care of patients with AML is virtually zero. Therefore, we set up strategies for the proteomic analysis of the mitochondria enriched cytoplasmic fractions in primary AML cells. Primary AML cells and normal blood cells were derived from bone marrow aspirates of a group of five patients with AML (M1 and M2) at the time of initial diagnosis before chemotherapy and three healthy controls. Peptide mass spectra were obtained on a MALDI-TOF-TOF mass spectrometer. Protein identification was processed and analyzed by searching the Swiss-Prot protein database using the MASCOT search engine of Matrix Science that integrated in the Global ProteinServer Workstation. Tandem electrospray ionization mass spectrometry (ESI-MS/MS) experiments were performed on a quadrupole (Q)-time of flight 2 (Q-TOF2) hybrid quadrupole/TOF mass spectrometry. We identified 48 spots corresponding to 38 proteins in primary AML cells, the expressions of which were altered significantly compared with normal blood cells. Out of these deregulated proteins, totally 12 and 20 proteins were observed in up- or down-regulated spots, respectively. Interestingly, prohibitin (gi4505773) was highly expressed in primary AML cells. Prohibitin is known to induce growth suppression and repress E2F-mediated transcription. But it has recently found that prohibitin protects cells from apoptosis by down-regulating E2F activity when Rb family members are inactive (Oncogene2002;21:4539–48). The current proteomic analysis for primary AML cells supports that prohibitin not only acts as a repressor of E2F activity during normal cellular conditions, but may also aid in the decision between survival and death during times of apoptotic stimulation. Our ongoing studies will try to elucidate the precise role and mechanism in primary AML cells. Six proteins (ADP-ribosylation factor guanine nucleotide-exchange factor 2, cytokeratin-9, vimentin, chain A, kinesin family member 1C and gi34533624 unnamed protein product) were exclusively identified in primary cells. In conclusion, this mitochondrial proteomic analyses enable us to define the deregulated mitochondria-related proteins important for the initiation and the progression of AML. Also, these analyses may promote the identification of new targets for specific treatment approaches in AML.
Vitamin E Isoforms Inhibit Cell Proliferation and Downregulate Homeobox Protein Expression in the Leukemic KG-1 Cells.
Authors: Saba Nakhle S. , Hammad Ahmad , Salman Huda , Campbell Sharon , Krishnan Koyamangalath
Published: October 2019

Vitamin E is a fat-soluble vitamin that exists in eight different isoforms: four tocopherols (alpha, beta-, gamma- and delta-), and four tocotrienols (also alpha-, beta-, gamma- and delta-). Studies are underway to determine whether vitamin E, through its ability to limit production of free radicals, might help prevent or delay the development of cancer. Other studies suggest that vitamin E may modulate signal transduction pathways to prevent or reverse the effects of cancer. In our study, we examined the effect of vitamin E isotypes on some gene products involved in acute leukemia such as HOXA9 (Homeobox A9), PBX1 (Pre-B cell leukemia transcription factor 1) and E2A-PBX1. HOXA9 is a transcription factor with a central role in both hemopoiesis and leukemia. A high level of HOXA9 is a characteristic feature of acute myeloid leukemia (AML) and may be sufficient to cause this disease. PBX1 is involved in a form of pre-B-cell acute lymphoblastic leukemia (B-ALL). E2A-PBX1 is a protein resulting from a chromosomal translocation t(1;19)(q23;p13.3) which involves PBX1 and E2A genes. E2A-PBX1 transforms cells by constitutively activating transcription of genes regulated by PBX1 or by other members of the PBX protein family. KG-1 cells were treated with 20 micromolar (μM) gamma tocotrienol (γT3) and delta tocotrienol (δT3) for 48 hours, and 50 μM gamma tocopherol (γT) and delta tocopherol (δT) for 72 hours. MTT Assays demonstrate 30% inhibition of cell proliferation using 50 μM γT after 72 hours, 60% inhibition of cell proliferation using 50 μM δT after 48 hours, 70% inhibition of cell proliferation using 25 μM γT3 after 48 hours, and 75% inhibition of cell proliferation using 25 μM δT3 after 48 hours. Western Blot analysis was performed on the cell lysates using HOXA9, PBX1 and E2A-PBX-1 antibodies. Our results showed that both δT and γT when used at a 50 μM concentration for 72 hours treatment, as well as δT3 and γT3 when used at a 20 μM concentration for 48 hours treatment, have a down regulatory effect on HOXA9, PBX1 and E2A-PBX-1 protein expression in the leukemic KG-1 cells. A down regulatory effect on cyclin D1 and cyclin D3 was also demonstrated.
Antitumor Activity of Bortezomib Alone and in Combination with TRAIL in Human Acute Myeloid Leukemia.
Authors: Conticello Concetta , Adamo Luana , Vicari Luisa , Giuffrida Raffaella , Iannolo Gioacchin , Anastasi Gabriele , Palumbo Giuseppe A. , Gulisano Massimo , De Maria Ruggero , Giustolisi Rosario , Di Raimondo Francesco
Published: October 2019

Acute myeloid leukemia (AML) is a malignant disease characterized by abnormal proliferation of clonal precursor cells. Although different strategies have been adopted to obtain complete remission, the disease actually progresses in about 60–70% of patients. Bortezomib has documented antitumor activity in multiple myeloma and other lymphoid malignancies. TRAIL is a member of the TNF family that induces apoptosis in tumor cells while sparing normal cells. Here we examined the sensitivity to Bortezomib alone or in combination with TRAIL of bone marrow cells from AML patients (34 patients: 25 newly-diagnosed, 4 relapsed, 5 refractory). Immunofluorescence analysis showed that NF-κB was located in the nuclei of the AML blasts and it did not translocate after exposure to Bortezomib. Bortezomib induced cell death in blasts from each patient sample. The cytotoxic effect was dose- and time-dependent (concentration from 0.001 to 10 μM for 24 and 48 hours) and was associated with down-regulation of Bcl-xL and Mcl-1, up-regulation of TRAIL-R1, TRAIL-R2, p21 and activation of executioner caspases. Moreover, low doses of Bortezomib primed TRAIL-resistant AML cells for enhanced TRAIL-mediated killing. Thus, the combination of proteasome inhibitors and TRAIL is effective for treating AML patients, even patients who are refractory to conventional chemotherapy.
The Production of Vascular Endothelial Growth Factor Is Decreased in Acute Myelogenous Leukemia Cases That Are Treated with Chemotherapeutic Agents and Show the Prolonged Bone Marrow Suppression.
Authors: Tashiro Haruko , Noguchi Mitsuho , Shirasaki Ryosuke , Kawasugi Kazuo , Shirafuji Naoki
Published: October 2019

Objective: There have been reported that the levels of serum vascular endothelial growth factor (VEGF) were decreased in aplastic anemia cases. We investigated VEGF system after chemotherapy to acute myelogenous leukemia (AML) cases, and determined whether VEGF system influenced the prolonged bone marrow suppression in these cases.

Materials and Methods: Sera and bone marrow cells were prepared from 30 AML cases including 10 cases of AML (M3) at the onset of the disease, after chemotherapy, and the recovery periods, and the concentration of VEGF in sera of the patients and in the conditioned media obtained from bone marrow-cell cultures was measured with ELISA kit (Quantikine; R&D Systems). The expression of VEGF, VEGF receptor type-1 and VEGF receptor type-2 was analyzed with RT-PCR. The biological effect of VEGF on the bone marrow cells which showed the prolonged suppression after chemotherapy was assayed with colony-formation with or without any cytokines.

Result and Discussion: As was reported previously, VEGF levels were significantly increased in M3 cases. In other types of AML cases the levels of VEGF production varied. When patients were given chemotherapy and the bone marrow suppression was prolonged, the production levels of VEGF were significantly diminished less than that observed in AML cases with normal bone marrow recovery. In M3 cases that were treated with all-trans retinoic acid and the prolonged bone marrow-suppression was observed, VEGF production was also suppressed. The expression of VEGFR-1 and -2 was observed in bone marrow cells from prolonged bone marrow suppression cases. In these cases, when bone marrow cells were cultured with VEGF, synergistic effects with G-CSF and EPO were observed with colony-formation assay. These observations indicate that VEGF works on the important role for the hematopoietic recovery after chemotherapy in AML cases.