bluuebunny/crossref_metadata_2025 huggingface dataset:
A Novel Fusion, KIAA0379 (Ankyrin Repeat Domain 28) to NUP98 in an Adult MDS/AML with a t(3;5;11)(p25;q35;p15).
Authors: Ishikawa Maho , Yagasaki Fumiharu , Okamura Daisuke , Maeda Tomoya , Sugahara Yuichi , Misumi Motohiro , Yoshida Katsuhiko , Nakamura Yuichi , Kawai Nobutaka , Matsuda Akira , Jinnai Itsuro , Bessho Masami
Published: April 2020

In hematologic malignancies, deletion of 3p25-26 is a rare but recurrent cytogenetic aberration, indicating the presence of tumor suppressor gene (TSG) on this choromosome arm.

We revealed a t(3;5)(p25;q35) that was identified by conventional cytogenetics results in a novel fusion of KIAA0379 on 3p25 to NUP98 on 11p15 in a patient with refractory MDS/AML by the molecular analysis. Fluorescence in situ hybridization (FISH) analysis using whole chromosomal painting and telomeric probes unclosed a criptic three-way translocation t(3;5;11)(p25;q35;p15). FISH restriction using BAC contig revealed that the 3p25 breakpoint is involved in KIAA0379 which locates between BAC 792N12 and 259O19. Split signals of BAC 265K23 for 5q35 and 348A20 for 11p15 showed that NSD1 on 5q35 and NUP98 on 11p15 are the responsible genes of this translocation. As the results of this translocation, expression of three chimeric transcripts were identified by RT-PCR. In KIAA0379-NUP98, exon 18 of KIAA0379 was fused in-flame with exon 13 of NUP98. In NUP98-NSD1, exon 12 of NUP98 was fused in-flame with exon 7 of NSD1. NSD1-KIAA0379 resulted in out-flame fusion. To investigate the expression of KIAA0379 in leukemic cells, RT-PCR for full-length KIAA0379 transcript was performed on bone marrow cells from the patient and a healthy volunteer, and leukemic cell line K562, HL-60 and KG-1a cells. Full-lengh PCR product with expected size was found in all samples, however, short variant transcript was amplified significantly from KG-1a and HL-60 cells. Subsequent sequence analysis of the short PCR product from KG-1a cells revealed a direct fusion of a part of exon 10 to a part of exon 28 without any hidden splicing sites. Southern blot analysis using a cloned KIAA0379 cDNA fragment suggested an internal deletion of KIAA0379 in KG-1a cells. KIAA0379 locates at 3p25.1 about 5.5Mb centromeric to VHL and is predicted to have 28 ankyrin repeat motifs by computational analysis. It is thought to be involved in protein to protein interaction whereas its precise funtion remains unclear. Immunostaining for KIAA0379 protein in NIH3T3 cells transfected with the wild KIAA0379 and the short variant KIAA0379 revealed that the wild KIAA0379 protein localized in cytoplasm and nuclear membrane, on the other hand, the truncated KIAA0379 protein localized exclusively on nuclear membrane. KIAA0379-NUP98 fusion protein is expected to localize on nuclear membrane by sub-localization signal of C-terminal of NUP98. Therefore, dysfunction of KIAA0379 protein might lead to leukemogenesis and KIAA0379 may be potential candidate for a novel TSG. In addition, it has been reported that t(5;11)(q35;p15.5) results in NUP98-NSD1 fusion in childhood AML. To our knowledge, this is the first report in adult case.
Experimental Design & Analysis of Protein Array Data: Applying Methods from cDNA Arrays.
Authors: Eckel Jeanette E. , Hoering Antje , Ghobrial Irene
Published: April 2020

It appears that a number of recent manuscripts using protein microarray technology are using equivalent analysis procedures that the gene-expression microarray community implemented in their infancy. That is, utilizing a classic reference design such that the ratio of the sample of interest to a reference sample is the response of interest and assessing fold change to determine differential expression. For example, recent publications have concluded that proteins with a fold change less than 0.7 or greater than 1.3 demonstrate significant down- or up-regulated differential expression, respectively. However, fold change is an unreliable measure of differential expression and statistical models that distinguish true signal from random noise should be utilized instead of fold changes. Over the last half decade a tremendous amount of research has been devoted to gene-expression microarrays to vastly improve on the areas of experimental design, normalization and statistical analyses to assess differential expression and classification and these methods are directly applicable to protein microarray technology. Thus, the objective is to review the statistical methodology that has been developed for two-color cDNA arrays that is directly applicable to protein arrays. Examples are provided from a mantle-cell lymphoma protein-array experiment.
Expression of Human Enhancer of Invasion 10 (HEI10) in Human Myeloid Leukemias and Downregulation during Differentiation of Hematopoietic Stem Cells.
Authors: Fujishima Naohito , Hirokawa Makoto , Fujishima Masumi , Ichikawa Yoshikazu , Horiuchi Takahiro , Miura Ikuo , Sawada Ken-ichi
Published: April 2020

Background: We previously investigated the gene expression profile of drug-resistant human leukemia K562 cells by the serial analysis of gene expression (SAGE) (Ichikawa et al. Int J Hematol, 2004;79:276), and found that the HEI10 gene was overexpressed in K562 cells. With the same technology, we also observed that human CD34+ cells expressed high levels of HEI10 gene expression compared to erythroid progenitor cells (Fujishima et al. Int J Hematol, in press). HEI10 encodes a protein which belongs to a family of E3 ubiquitin ligase,and interacts with cyclin B1 leading to the regulation of cell cycle. In the present study, we examined the gene expression of HEI10 quantitatively in human leukemias in order to learn whether there would be any association between the expression levels and leukemia subtypes. We also investigated whether the expression levels of HEI10 would be related to differentiation of myeloid leukemia cell lines and normal hematopoietic stem cells.

Methods: The quantitative real-time PCR method was employed to estimate the relative gene expression levels of HEI10. Total RNA was extracted from mononuclear cells of bone marrow that had been taken for diagnostic purposes. Patients include acute myeloid leukemia (AML) (n=21) and chronic myeloid leukemia (CML) (n=7; 2 in blastic crisis, 5 in chronic phase). Differentiation of human acute myeloid leukemia cell line HL60 cells was induced by PMA. Human normal CD34+ cells were purified from G-CSF mobilized blood stem cells and were induced to differentiate into erythroid, myeloid or dendritic cells by various cytokines.

Results: Quantitative real-time PCR analysis revealed the high expression of HEI10 gene in patients with leukemia with undifferentiated phenotype (3 cases of AML M1 in FAB classification and 2 cases of CML in blast crisis) and low levels of expression in acute promyelocytic leukemia and CML in chronic phase. When HL60 cells were induced to differentiation with PMA, accumulation in the G0/G1 phase was observed and the expression of HEI10 was decreased. Differentiation of normal CD34+ cells also resulted in a decrease of HEI10 expression.

Conclusions: The expression of HEI10 gene in hematopoietic cells appears to be upregulated when cells are immature. The role for HEI10 in hematopoietic stem cells remains to be elucidated. Biological effects of the HEI10 gene silencing in human hematopoietic cells are currently being investigated by RNAi technology.

Figure Figure Figure Figure
Identification of TAL1/SCL Target Genes through siRNA and Microarray Expression Analysis.
Authors: O’Neil Jennifer , Ferrando Adolfo A. , Palomero Teresa , Look A. Thomas
Published: April 2020

TAL1 is a basic helix-loop-helix (bHLH) protein that is essential for hematopoiesis. Activation of the TAL1/SCL gene occurs in as many as 60% of patients with pediatric and adult T cell acute lymphoblastic leukemia (T-ALL). Mouse models have demonstrated that TAL1 transforms by inhibiting the function of E proteins, however the transcriptional targets of TAL1 that contribute to leukemia are not known. In this study we sought to identify targets of TAL1 that contribute to the transformation of thymocytes through siRNA-mediated knockdown of TAL1 expression in the Jurkat T-ALL cell line that expresses high levels of TAL1. We have generated several clonal cell lines that stably express a siRNA directed against TAL1 with up to 90% knockdown of TAL1 at both the RNA and protein level. Phenotypic characterization of these cells revealed that Jurkat cells with decreased TAL1 expression display a decreased proliferative capacity. This phenotype is augmented in lower serum concentrations in which wild-type and siRNA control Jurkat cells proliferate rapidly. Microarray expression analysis using Affymetrix U133 Plus 2.0 arrays has revealed that TAL1 is a global regulator of gene expression and that many pathways are affected by TAL1 knockdown, including those implicated in cell growth and proliferation. This data will enable us to dissect the downstream pathways of TAL1 by knocking-down or overexpressing these TAL1 targets (depending on whether they are up- or down-regulated in the absence of TAL1), and assessing whether they affect the proliferative rate or survival T-ALL cells. We anticipate that we will identify and validate potential therapeutic targets for TAL1-induced leukemia using this combination of approaches.
Genetic Susceptibility to Hodgkin Lymphoma.
Authors: Cozen Wendy , Salam Muhammad T. , Nieters Alexandra , Ingles Sue A. , Mack Thomas M. , Masood Rizwan , Cockburn Myles G. , Gauderman William J. , Wang Wei , Gill Parkash S.
Published: April 2020

A case-control study of young adult Hodgkin lymphoma (diagnosed before age 50) in twins was conducted to determine if cases and their unaffected identical twins demonstrated dominance of Th1, Th2 or regulatory cytokines compared to unrelated controls. We collected blood specimens from sets of young adult Hodgkin lymphoma cases (n=60), their unaffected identical twins (n=60) and unrelated controls (n=60) and measured IL-6, IL-8, IL-10, and IL-12 levels in PHA-stimulated PBMC supernatant by ELISA. IL-6, IL-10 and IL-12 promoter polymorphisms were determined using PCR and either Taqman or Pyrosequencing. Mean levels of cytokines were compared using an analysis of covariance and genotypes were compared using multivariate logistic regression (SAS). Both cases and their unaffected twins had higher levels of IL-8, but lower levels of IL-12, compared to controls. Unaffected twins of cases had IL-6 levels that were 86% higher than those of controls (p=0.02), however cases’ levels were lower than controls (altered by treatment). Cases and their twins were three times more likely to have an IL-6 genotype associated with higher levels and 2.6 times more likely to have an IL-12 genotype associated with lower levels, compared to controls (Table 2). There were no significant differences in IL-10 levels or genotypes between cases and controls. A genetically determined skewing toward increased inflammatory cytokines (IL-6 and IL-8) and decreased Th1 cytokines (IL-12) is associated with increased susceptibility to young adult Hodgkin lymphoma.

Table 1. Mean and % Difference in Cytokine Levels in Hodgkin Lymphoma Cases, their Unaffected Identical Twins, and Controls

Hodgkin Lymphoma Case v. Control Unaffected Twin of Case v. Control Cytokine Mean Diff (pg/ml) % Diff P-Value Mean Diff (pg/ml) % Diff P-Value IL6 −43.2 −8.7 0.75 +372.8 +83.6 0.04 IL8 +1978.7 +42.9 0.36 +1342.3 +61.9 0.26 IL10 −3.9 −3.0 0.85 −25.6 −20.4 0.20 IL12 −1.2 −54.8 0.01 −0.7 −32.0 0.27

Table 2. Cytokine Genotype and Risk of Young Adult Hodgkin Lymphoma in Cases v. Controls

Cytokine Gene SNP Genotype Odds Ratio 95% Confidence Interval *reference genotype IL6 −174 G>C GG v. CC* 3.12 1.03–9.44 IL12 +1188 A>C CC v. AA* 2.59 1.03–6.55
Population Based Genotyping of Human Leukocyte Antigen (HLA) in Hodgkin Lymphoma: EBV Positive HL Is Associated with HLA Class I and EBV Negative HL Is Associated with HLA Class III.
Authors: Diepstra Arjan , Niens Marijke , Vellenga Edo , te Meerman Gerard J. , Schaapveld Michael , Nolte Ilja M. , van Imhoff Gustaaf W. , Poppema Sibrand
Published: April 2020

An inherited susceptibility to Hodgkin Lymphoma (HL) is indicated by the variation in incidence among race/ethnicity groups, aggregation of the disease in families and by association with human leukocyte antigens (HLA). In this study we conducted a detailed screening analysis of the complete HLA region in EBV-positive and EBV-negative patients with HL.

In total, 200 patients and 348 family-based controls of the Caucasian population of the Northern Netherlands participated in this population-based study. Paraffin embedded tissue of all patients was obtained and used for revision according to the WHO classification. In addition, analysis of EBV status was performed. 54 patients were EBV-positive (33 NS, 14 MC, and 7 other classical cases) and 146 patients were EBV-negative (124 NS, 4 MC, 13 NLP, and 5 other classical cases). 33 microsatellite markers located in the HLA region 6p21 were used for genotype screening.

Allele, genotype and two-locus haplotype association analyses were used to search for differences between EBV-positive and negative patients and family controls using a t-test to assess significance. In the EBV-positive patient group, HL was associated with an allele of 126 bp for D6S265 and an allele of 284 bp for D6S510 (p=0.00031). These markers are located in the HLA class I subregion. Individuals homozygous for 126 allele of D6S265 were observed in 34.4% of the EBV-positive patient group and 5.2% of controls (odds ratio (OR)= 6.6). The odds ratio for individuals heterozygous for 126 allele of D6S265 was lower (OR= 2.5).

In addition, Haplotype Sharing Statistics (HSS) was used to search for differences in haplotype sharing between patients and family controls. The HSS assumes that haplotype segments of patients from a founder population are conserved in the region spanning a disease locus.

The HSS analysis showed strongest haplotype sharing around marker D6S273 in patients compared to controls (p<0.0001). The sharing was less evident in the EBV-positive subgroup compared to the EBV-negative subgroup, possibly due to low power. D6S273 is located at the telomeric end of the HLA class III region nearby TNF-alpha, TNF-beta and heat shock protein genes. Our results indicate that part of the HLA class I and part of the HLA class III region are associated with susceptibility to classical HL, with the HLA class I association being specific for EBV positive HL. We will further analyze both regions aiming to find genes and polymorphisms associated with the development of HL.
Mutations of the PTPN11 Gene in Childhood Hematological Malignancies.
Authors: Chen Yuyan , Takita Junko , Hiwatari Mitsuteru , Hanada Ryoji , Kikuchi Akira , Tsuchida Masahiro , Sugita Kenichi , Sako Masahiro , Taki Tomohiko , Ogasawara Mizuho , Hayashi Yasuhide
Published: April 2020

The PTPN11 gene encodes SHP2, which is a protein tyrosine phosphatase functioning as signal transducer downstream to growth factors and cytokine receptors and its function is mediated, at least in part, through the Ras/Raf/ERK cascade in hematopoietic and non-hematopoietic cells. Recently, it has been reported that germline mutations in PTPN11 cause Noonan syndrome and a gain-of-function somatic mutations in PTPN11 has also been reported in juvenile myelomonocytic leukemia (JMML). To investigate the prevalence of mutations in PTPN11 in childhood acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL), we screened the hot spot of PTPN11, exon 3, in 80 cell lines, including 14 AML, 5 chronic myeloid leukemia, 14 T-cell ALL and 47 B-precursor ALL cell lines, and 207 fresh samples, including 20 myelodysplastic syndrome (MDS), 3 JMML, 85 AML, 57 B-precursor ALL patients as well as 42 infant leukemia patients (6 AMLs, 35 ALLs, 1 mixed lineage leukemia), using polymerase chain reaction-single strand conformation polymorphism and direct sequencing analyses. In exon 3, mutations were observed in 2 of 14 (14.3%) AML cell lines (Asp61Val and Glu76Gly). A missense mutation (Ala72Val) was detected in 1 of 85 (1.2%) AML patients (a 9-year-old girl with FAB-M0). In 20 MDSs, a missense mutation (Asp61Val) (5%) was identified in a RAEB-T patient. In 3 JMMLs, 2 missense mutations (66.7%) were detected; one was Gly60Ala, and the other was Glu76Val. In 42 infant leukemia patients, we detected only one missense mutation (Ala72Asp) (2.4%) in an AML-M5b patient. However, we did not detect any mutations of exon 3 in 61 ALL cell lines and 92 ALL patients including 35 infant ALL patients. The screening for other coding regions is still undergoing although more than 90% of the reported mutations were clustered in exon 3. In conclusion, our data suggest that the PTPN11 mutations are involved in myeloid hematological malignancies rather than ALL.
Expression of Cyclooxygenase-2 (COX-2) in Human Leukemias and Hematopoietic Cells.
Authors: Lilly Michael B. , Drapiza Leslie , Sheth Milan , Zemskova Marina , Bashkirova Svetlana , Morris Joan
Published: April 2020

COX-2 has been implicated in the development of many epithelial cancers, as well as in tumor angiogenesis. COX-2 inhibitors have been shown to have anti-tumor activity in experimental cancer. Little information exists, however, on the expression or role of COX-2 in hematologic malignancies. We have use a variety of immunochemical assays to document expression of COX-2 in human and murine leukemias and hematopoietic cells. The factor-dependent murine cell lines FDCP1 and 32D expressed COX-2 when growing continuously in the presence of IL-3; expression declined markedly when growth factor was removed. FDCP1 cells constitutively expressing bcl-2, pim-1, or bcr-abl had markedly elevated levels of COX-2, and continued to express this enzyme even after removal of growth factor. To assess COX-2 expression in human hematopoietic cells we developed a flow cytometry assay using a FITC-labelled anti-COX-2 MoAb (Cayman). Cells were washed once in serum-free medium, fixed briefly in 1% paraformaldehyde, permeabilized with PBS/0.2% Triton X100, then stained with antibody. Negative control samples were processed similarly but stained with antibody that had been preincubated with immunizing peptide. Specific COX-2 staining was interpreted as the difference between the histograms from blocked versus unblocked anti-COX-2 antibody, as determined by Kolmogorov-Smirnoff analysis. In buffy coat preparations from normal donors, we found constitutive expression of COX-2 in lymphocytes (both B-cells and T-cells). In contrast little or no COX-2 was detected in unstimulated neutrophils or monocytes. In human acute myelogenous leukemia (AML) cell lines we found COX-2 expression to be universal and easily detected. In several cell lines we confirmed the results of our flow cytometry assay with immunoblotting. We further examined 25 cryopreserved samples of human acute leukemia blasts obtained from peripheral blood. COX-2 expression was variable, but universal. Levels generally were less than those seen in immortalized cell lines, and did not correlate with blasts morphology (AML, ALL, APL, AMoL, CML-BT). To determine if COX-2 inhibitors could play a role in the treatment of acute leukemias, we performed cytotoxicity assays using the COX-2 specific inhibitors, celecoxib and NS398. Survival and growth of human AML cell lines were inhibited by both agents. These data demonstrate that 1) a variety of oncogenes can induce expression of COX-2 in hematopoietic cells, 2) clinical human acute leukemias uniformly express COX-2 in circulating blasts, and 3) COX-2 inhibitors are cytotoxic for human leukemia cells. Combination therapies for acute leukemias may evaluate the incorporation of COX-2 inhibitors for added cytotoxic effects or angiogenesis inhibition.
Characterization of Secondary Alterations in Mantle Cell Lymphomas Using Matrix-/Array CGH.
Authors: Steinemann Doris , Tauscher Marcel , Rudolph Cornelia , Emura Makito , Abe M. , Daibata M. , Matsuo Yoshinobu , Drexler Hans G. , Radlwimmer Bernhard , Kestler Hans A. , Lichter Peter , Schlegelberger Brigitte
Published: April 2020

Overexpression of Cyclin D1 due to the chromosomal translocation t(11;14)(q13;q32) alone is not sufficient to induce the development of mantle cell lymphoma (MCL). Therefore, secondary genetic alterations seem to be necessary for the malignant transformation and may determine the clinical course of the disease. In order to define chromosomal imbalances more precisely, we analyzed six t(11;14)-positive mantle cell lymphoma cell lines (Granta-519, NCEB1, JeKo-1, Rec-1, SP-53 and HBL-2) by means of matrix-/array-based comparative genomic hybridization (array-CGH), a new molecular tool that allows the genome-wide screening of chromosomal imbalances. The microarray used contains 6251 individual BAC/PAC clones. Clones were selected to reach a genome-wide resolution of 1 Mb and an even higher resolution of up to 100kb in recurrently aberrant regions of MCL. In addition, regions containing known tumor suppressor genes and oncogenes were covered by a high density of clones. The hybridization of tumor DNA against normal control DNA and the subsequent washing steps were carried out using the automated slide processor Lucidea (Amersham-Pharmacia Biotech). Based on the normalized fluorescence ratios computed as log2 values, we were able to detect amplifications as well as single gains and losses. The most frequent alterations were losses in 9p21.3, 2p11.2, 22q11.22, 1p21.1-p31.2, 13q14, 11q22, 6q21, 6q27, 17p13 and gains in 7p14.1, 7q11.2, 2q37.1, 8q24, 12q13 and 18q21. These results are in agreement with the recently published data from deLeeuw et al. (HMG Advance Access June 30, 2004). Up to now, losses of 2p11.22 (all cell lines except for JeKo-1) and 22q11.22 (all cell lines except for Rec-1) have not been known as recurrent aberrations of MCL. Selected chromosomal losses (p16, p53) and the amplification of BCL2 in Granta-519 were confirmed by means of fluorescence in situ hybridization using commercially available probes (Abbott Diagnostics, Wiesbaden, Germany). Matrix-/array CGH analyses enabled us to further delineate important regions of gain and loss like 13q14. In conclusion, the cell lines are excellent model systems which will facilitate the identification and characterization of novel genes which play important roles in the pathobiology of MCL.
Rare Mutations of the PIK3CA Gene in Hematologic Malignancies.
Authors: Muller Claudia I. , Miller Carl W. , Hofmann Wolf-K. , Kawamata Norihiko , Koeffler H. Phillip
Published: April 2020

Phosphatidylinositol 3-kinase (PI3K) is a lipid kinase that is activated in advanced cancers through inactivation of the PTEN tumor suppressor gene. Somatic mutations in the p110-alpha catalytic subunit, PIK3CA occur in colon, gastric, breast and lung cancers as well as in glioblastomas and colorectal adenomas. Mutations were found in two “hot spots”: the helical (exon 9) and kinase (exon 20) domains. Mutational analysis of PIK3CA in hematologic malignancies has not been reported. We performed a screening analysis of 28 cell lines from human hematologic malignancies as well as 46 AML and MDS samples and 42 lymphoma samples by polymerase chain reaction - single strand conformation polymorphism (PCR-SSCP) and nucleotide sequencing of the two “hot spots”. Two colon adenocarcinoma cell lines (SKCol1, SW403) were used as positive controls and revealed the known missense mutation (Q546K) in exon 9. A novel missense mutation occurred in the erythroid chronic myeloid leukemia cell line K562, causing an amino acid change from Glutamic acid to Alanine at amino acid position 545 of exon 9 (E545A). In the kinase domain (exon 20), a novel polymorphism was observed in two different MDS patients at Threonine residue 1025. Experiments are exploring the functional importance of the PIK3CA mutation and the PIK3CA polymorphism in exon 20. In summary, PIK3CA mutations are infrequent in hematologic malignancies.
Identification of Target Genes for the Tumor Suppressor RIZ1 Using Gene Expression Profiling.
Authors: Dong Wei-Feng , Takahashi Naoto , Bainbridge Matthew N. , Hull Andrea R. , Scott Stuart A. , Geyer C. R. , DeCoteau John F.
Published: April 2020

RIZ1 (PRDM2) is a tumor suppressor gene on 1p36 that frequently undergoes deletion, rearrangements, and loss of heterozygosity in a broad spectrum of tumors. RIZ1 is a member of the nuclear protein methyltransferase superfamily involved in chromatin remodeling. RIZ1 contains a ~130 amino acid conserved domain (PR or SET) that is important in chromatin-mediated regulation of gene expression and in the development of cancer. RIZ1 methylates Histone H3 on K9 and this activity may play a role in transcription repression as H3-K9 methylation is known to be associated with repression. Aberrant activities or mistargeting of chromatin modifying activities are proving to have unexpected links to cancer. We and others have shown that RIZ1 expression is down regulated in human leukemias and in the human erythroleukemia cell line K562. Expression of RIZ1 in K562 reduced proliferation, increased apoptosis, and promoted erythroid differentiation. To understand how RIZ1’s DNA binding, methyltransferase, and transcription repressor functions are related to its tumor suppressor activity it is necessary to characterize RIZ1 target genes. We used DNA microarrays to globally monitor how RIZ1 affects gene expression profiles. We constructed a K562 cell line with RIZ1 stably integrated under the control of a CMV promoter and analyzed the gene expression profiles of K652 and K562 + RIZ1 using a 42K Stanford human gene microarray. By comparing the gene expression profiles of these cell lines, we identified potential RIZ1 gene targets that are up and down regulated in the presence of RIZ1. In total, we identified 5 upregulated genes and 20 down regulated genes using significance analysis of microarrays (SAM) and standard deviation filter analysis of the gene expression data. RIZ1-mediated changes in gene expression profiling indicate that RIZ1 is potentially involved in the regulation and connection of the IGF-1 (IGF-1, IGFBP2) and integrin (LMS1) pathways, and in the activation of the TGF-β (SPARC) pathway. The genes perturbed by RIZ1 expression suggest that the tumor suppressor properties of RIZ1 arise from its control of proliferation, apoptosis and differentiation using these pathways. Finally, we observed an overrepresentation of the SP-1 transcription factor binding sites in genes that are upregulated in the absence of RIZ1. This correlates with the ability of RIZ1 to recognize SP1 sequences.
Effect of Dioxin on Normal Human Hematopoietic and Leukemic Cells.
Authors: Deliliers Giorgio Lambertenghi , Fracchiolla Nicola S. , Servida Federica , Bosari Silvano , Soligo Davide
Published: April 2020

2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is the prototypical agonist of the aryl hydrocarbon receptor (AhR), a member of the erb-A family that also includes receptors for steroids, thyroid hormones, peroxisome proliferators and retinoids. When bound to dioxin, the AhR can bind to DNA and alter the expression of various genes, including cytokines and growth factors. TCDD has a large number of biological effects, such as long-lasting skin disease, cardiovascular disease, diabete and cancer. An increase risk of Hodgkin’s disease, non-Hodgkin’s lymphoma and myeloid leukemia was observed after 15 years in the population exposed to dioxin following the 1976 accident in Seveso, Italy. In this study, we analysed the effect of escalating doses of TCDD on the human myeloid leukemic cell lines HL60 (promyelocytic leukemia) and K562 (chronic myelogenous leukemia), as well as on human CD34+ progenitor cells from the leukapheresis of normal donors stimulated with G-CSF. The possible specific modulation of gene expression induced by TCDD was then tested by means of macroarray analyses (Clontech), the results of which were successfully validated by real-time quantitative PCR on five candidate genes. The dioxin dose capable of inhibiting the growth of 50% of the colonies in semisolid medium (IC50 calculated using the CalcuSyn computer program) was 33 nM for HL60, 28 nM for K562, and 20 nM for the CD34+ cells. The HL60, K562 and CD34+ cells tested before and after exposure to 20 nM of TCDD allowed us to identify a series of modulated genes. The up-regulated genes included LUCA15 putative tumour suppressor, cyclin-D binding Myb-like protein (hDMP1), MAPK/ERK kinase 6, ras-related protein RAB2, RalB GTP-binding protein, transcription factor ZFM1, and BRCA1-associated ring domain protein (BARD1). The down-regulated genes included found c-myc, TNF alpha precursor, ICAM1, MIP1-beta, interferon gamma-induced protein precursor (gamma-IP10), granulocyte-macrophage colony stimulating factor (GM-CSF), monocyte chemotactic protein 1 precursor (MCP1). All of these genes are variously involved in the processes of proliferation, differentiation and transformation in hematological and tumoral cell models. On the basis of these data, we can hypothesise that the inhibition of clonogenic potential and the gene expression induced by TCDD exposure on normal CD34+ progenitor cells plays a role in the neoplastic transformation of hemopoietic stem cells. This supports the epidemiological data indicating an increased hematological cancer risk in the population accidentally exposed to TCDD.
Proteinated Elemental Selenium Potentiates Anti-Tumor Effect of Ionizing Radiation and Chemotherapeutic Agents but Is Only Minimally Affected by Drug Resistance Mechanisms.
Authors: Tsujino Ichiro , Miyagi Kiyoko , Sampson Reynee W. , Gunther Wolfgang H.H. , Krieg Marianne , Sieber Fritz
Published: April 2020

Elemental selenium generated by the photobleaching of selone photosensitizers combined with serum albumin and lipoproteins to generate conjugates that were highly cytotoxic to leukemia and selected solid tumor cells but well tolerated by normal CD34-positive hematopoietic cells. Selenium-protein conjugates were internalized by an endocytotic mechanism and localized in lysosomes. Internalized conjugates caused an extremely rapid and extensive (up to 80% in 1 hour) depletion of intracellular glutathione (GSH), the oxidation of 2′,7′-dichlorofluorescin, a loss of plasma membrane asymmetry, a breakdown of mitochondrial potential, and the activation of several caspases. Additive or synergistic anti-tumor effects were obtained when Se-protein conjugates were used in combination with ionizing radiation, melphalan, amifostine, 4-hydroperoxycyclophosphamide, edelfosine, buthionine sulfoximine, or moderate doses of arsenic (III) oxide. For example, pretreatment with a nontoxic dose of amifostine enhanced the depletion of L1210 leukemia cells from 7 log to 12 log (depletions in excess of 6 log estimated from linear extrapolations of survival curves) while the yield of normal CD34-positive cells remained at 100%. Antagonistic interactions were obtained with simultaneously applied cisplatin or very high doses of arsenic (III) oxide. The antagonistic interaction with cisplatin probably reflected a competition of cisplatin and selenium for the same binding site at or near cysteine-34 of albumin. Melphalan-resistant L1210/L-PAM1 leukemia cells (elevated intracellular GSH) were more sensitive to Se-protein conjugates than wild-type cells because of an unexpected increase in conjugate uptake by mutant cells. Cisplatin-resistant H69/CDDP cells (elevated GSH, metallothioneine, glutathione-S-transferase-pi) were slightly more sensitive than wild-type cells. Cisplatin-resistant PC14/CDDP cells (reduced drug uptake) and adriamycin-resistant HL-60/ADR cells (MRP-mediated efflux) were slightly less sensitive than the corresponding wild-type cells. In the latter cases, the reduced sensitivity of mutant tumor cells correlated with reduced conjugate uptake and/or higher intracellular GSH levels. Taken together, the above results suggest that compounds modeled after our Se-protein conjugates may prove useful for the systemic therapy of leukemia and selected solid tumors. Supported by CA77387, MACC Fund, Children’s Hospital Foundation, and Wisconsin Breast Cancer Showhouse for a Cure.
Blockade of Highly Expressed CD55 Related to Tumor Bulkiness by RNA Interference Overcomes Resistance to CDC with Rituximab.
Authors: Terui Yasuhito , Sakurai Takuma , Mishima Yuko , Mishima Yuji , Yokoyama Masahiro , Takahashi Shunji , Itoh Yoshinori , Hatake Kiyohiko
Published: April 2020

[Background] Recently, We have presented that sensitivity to anti-CD20 antibody-mediated CDC was different during cell growth dependent on expression of CD55 (ASH 2003, San Diego). In some cases with malignant lymphoma after the treatment of anti-CD20 antibody, lymphoma cells get resistance to the treatment of anti-CD20 antibody. Moreover, bulky disease of malignant lymphoma like 7.0 cm of tumor gets resistance to rituximab. One of some regulators in complement system, CD55, is a major regulator of the alternative and classical pathways of complement activation and is expressed on all serum-exposed cells. Here, we have found the relationship among extirpated lymph nodes, sensitivity to CDC, and CD55 expression, and small interference of RNA (siRNA) for CD55 is a useful tool for blockade of CD55 in monoclonal antibody therapy.

[Methods] Human B cell line, Daudi and Rajii cells were used as sensitive and insensitive controls in complement-mediated cytotoxycity (CDC) with anti-CD20 antibody. Lymphoma cells were collected from extirpated lymph nodes of the patients with lymphoma. CDC assay with anti-CD20 antibody was performed with FACscan or under confocal laser scanning microscopy system. Three parts of siRNAs for CD55 (CD55-N, -M, and -C) were designed, and transfected to the cells using lipofectin

[Results] The relationship between sensitivity to CDC and size of extirpated tumor showed negative correlation. The formula’s intercept was 7.775 cm, suggesting that lymphoma with more than 7 cm of size is not effective in rituximab therapy as Coiffier’s report. The level of CD55 expression on lymphoma cells was correlated with size of its lymph node, and in some patient with lymphoma, level of CD55 expression on Lymphoma cells and susceptibility to CDC were different among before, after relapse and after partial remission, indicating that susceptibility to CDC depends on CD55 expression. This suggests that increased cell numbers contribute to higher or enhanced CD55 expression and resistance to CDC with rituximab. Lymphoma cells from all five cases with recurrent lymphomas strongly expressed CD55 molecules under laser scanning confocal microscopy. Percentages of dead cells showed no significant differences among transfection with and without CD55-N, CD55-M and CD55-C before CDC assay. Percentage of PI-positive cells in transfection with CD55-N significantly increased from 7.1% to 67.9%. These indicate that the siRNA against CD55 (1–380 nucleotides), CD55-N could efficiently knock down the expression of CD55 on freshly isolated lymphoma cells from recurrent lymphomas, and that it could induce cell death in freshly isolated lymphoma cells from recurrent lymphomas by CDC.

[Conclusion] This siRNA for CD55 could be a good tool for blockade of CD55 in resistance and bulkiness related with highly expressed CD55 with monoclonal antibody therapy.
Induction of Apoptosis in K562 Human Chronic Myeloid Leukemia Cells by Tetra-Arsenic Tetra-Sulfide.
Authors: Ye Qidong , Gu Long-Jun , Zhao Yanxia , Zhao Jincai , Chen Wengao , Zhang Bei , Jiang Limin
Published: April 2020

Realgar has been used as a traditional medicine in China for more than 1500 years. Some studies found that tetra-arsenic tetra-sulfide (As4S4), the main ingredient of realgar, used alone was highly effective and safe for all stages of acute promyelocytic leukemia. To explore the effects of As4S4 in treatment of human chronic myeloid leukemia K562 cells, we used microculture MTS assay to measure the growth inhibition of K562 cells. The morphologic change was determined by Wright’s staining and Hoechst33342 assay. Cell apoptosis was evaluated by DNA agarose gel electrophoresis. The apoptotic rate and cell cycle were measured by flow cytometry. The changes of transcript and protein levels were determined by real-time reverse transcription-PCR and Western blot analysis, respectively. As4S4 had signigicant cytotoxicity on K-562 cells. At the concentration of 2.0μmol/L, the cell viability decreased significantly after 24 hours cultured with the reagent. When the concentration was lower than 0.5μmol/L, As4S4 had little effect on K562 cells. The effect of As4S4 on K562 was time and concentration dependent. After cultured with As4S4 at the concentration of 2.0μmol/L for 24 to 48 hours, K562 cells appeared typical morphological changes of apoptosis. At a concentration greater than or equal to 2.0μmol/L, As4S4 could induce apoptosis significantly. After 12 hours of incubation with 2.0μmol/L As4S4, the apoptosis rate increased from 2.05% to 12.03%. At the same time, the percentage of cells in G1 phase decreased from 69.65% to 50.53%, whereas the percentage of G2/M phase increased from 9.56% to 25.91%. The mRNA levels of BCL-2, BCL-XL, BAD and BAX, and the protein levels of Akt and pAkt down-regulated after the inhibition of As4S4. The transcript and protein levels of BCR-ABL had no change after incubation with As4S4. These results indicated that As4S4 can inhibit the growth of K562 cells efficiently through inducing apoptosis and cell cycle arrest. It seems that As4S4 interferes with Akt pathway and down-regulate BCL-2, BCL-XL, BAD and BAX, which may be involved in the response of K562 to this agent. As4S4 could be beneficial for treatment of CML in combination with conventional drugs.
The Protein Kinase C beta (PKCβ) Inhibitor Enzastaurin HCl (LY317615) Augments Chemosensitivity of Multiple Myeloma (MM) Cells to Dexamethasone and the Proteasome Inhibitor Bortezomib.
Authors: Suvannasankha Attaya , Xu Dawei , Abonour Rafat
Published: April 2020

PKCβ isoforms are important in B-cell signaling and proliferation. PKCβ overexpression in diffuse large B-cell lymphoma relates with poor prognosis but the clinical significance of PKCβ in MM is not yet established. Herein, we report antimyeloma activity of the PKCβ inhibitor enzastaurin HCl (LY317615) both as a single agent and in combination with bortezomib and dexamethasone.

Methods: Six MM cell lines (NCI-H929, RPMI8226, IM9, U266, ARH-77 and HS Sultan) with varying degree of PKC activity were treated with graded dose of enzastaurin and studied for induction of apoptosis by flow cytometric analysis of annexin and propidium iodide stainings, and inhibition of proliferation by MTT assay.

Results: IC50 doses of both effects were low at 1-5 μM in all cell lines and did not correlate with the baseline PKC activity. Interestingly, the combination of enzastaurin at non-toxic concentrations with varying doses of dexamethasone and bortezomib yielded synergistic enhancement of cell death and inhibition of proliferation in all cell lines. Multiparameter immunoblottings of cell lysate after enzastaurin treatment showed that, at these functional doses, PI3/AKT, Erk and JNK pathways were unaffected. Additional studies are ongoing to evaluate the effect of enzastaurin on activities of apoptotic proteins, and verify its effect in primary samples.

Conclusions: Our preliminary data indicate that the PKCβ inhibitor enzastaurin demonstrates single-agent antimyeloma activity and can sensitize MM cells to bortezomib and dexamethasone. Enzastaurin is promising for further studies as an approach to increase activity and decrease toxicity of MM therapy.

Figure Figure
MHH-TALL1, a T-ALL Cell Line Expressing a Novel BCR-ABL Transcript.
Authors: Quentmeier Hilmar , Cools Jan , MacLeod Roderick A.F. , Marynen Peter , Uphoff Cord C. , Drexler Hans G.
Published: April 2020

The Philadelphia chromosome (Ph) is the result of the chromosomal translocation t(9;22)(q34;q11), leading to the BCR-ABL fusion gene. The Ph chromosome is the hallmark of chronic myeloid leukemia, but is also detected in acute lymphoblastic leukemia (ALL), particularly in adults. The majority of Ph-positive ALL cases belong to the category of B-cell precursor ALL, whereas Ph-positive T-ALL cases are rather rare. The MHH-TALL1 cell line was established from the peripheral blood of an 11-year-old boy with T-ALL in 1993. PCR analysis of primary tumor cells failed to reveal the existence of a BCR-ABL fusion. Interestingly however, conventional cytogenetics and fluorescence in situ hybridization performed on the cell line showed a 3-way t(1;9;22)(q32;q34;q11) rearrangement effecting Ph formation. As with the patient, standard RT-PCR of the various known BCR-ABL fusion transcripts was negative in the cell line. However, a weak band, about 600 bp larger than the usual e1-a2 BCR-ABL transcript was detected, and subsequently confirmed on reanalysis after optimizing PCR conditions. Sequencing of the RT-PCR product showed that MHH-TALL1 expressed an e6-a2 BCR-ABL fusion transcript. Northern and Western blot analyses demonstrated that the BCR-ABL gene products were expressed at very low levels only. It may be speculated that the presence of this novel BCR-ABL variant has been overlooked in previous analyses, because (i) PCR conditions used to screen for BCR-ABL fusion transcripts were not optimal to detect this variant and (ii) a weak signal running at the “wrong” size might have been neglected. In summary, we report a novel BCR-ABL fusion variant expressed in a T-ALL cell line. Our data raise the intriguing possibility that some BCR-ABL negative cases may express the novel e6-a2 transcript described herein. This might have an impact on treatment of the respective patients with ABL kinase inhibitors.
Aberrant Expression of the Chondroitin Sulfate Proteoglycan NG2 on Pediatric AML/abn(11q23) and ALL/t(4;11) Does Not Allow Flow Cytometric Detection of Leukemic Blasts with a Stem Cell-Like Immunophenotype.
Authors: Neudenberger Janna , Hotfilder Marc , Rosemann Annegret , Langebrake Claudia , Reinhardt Dirk , Vormoor Josef
Published: April 2020

Monitoring of minimal residual disease in childhood leukemia is widely used to determine treatment response and to detect persisting leukemic blasts for relapse prediction. In this context, it is increasingly recognized that only few leukemic blasts possess self-renewal capability and that only these self-renewing leukemic stem cells are able to initiate relapses. Therefore, the leukemic stem cells as the relapse-initiating cells should be the target cells of minimal residual disease detection. The human homologue of the rat chondroitin sulfate proteoglycan NG2 protein (detected by the monoclonal antibody 7.1) is aberrantly expressed in a high percentage of leukemias harboring 11q23 abnormalities and its expression has been proposed as a potential marker for MRD detection. To evaluate NG2 as a marker for leukemic stem cells, bone marrow or peripheral blood samples of 4 AML/t(9;11), 1 AML/t(10;11), 1 AML/t(?;11) and 2 ALL/t(4;11) patients with NG2-positive leukemia were analyzed by four color flow cytometry for NG2 expression on the cell surface of primitive cell populations. Immature cells were defined by the antigen profiles CD34+CD38− (CD33−) in AML and CD34+(CD117+)CD19− in ALL. AML stem cells are known to be CD34+CD38−. In the ALL patients, 32% resp. 81% of CD34+CD19− cells were leukemic by FISH analysis for MLL/AF4. For this rare event analysis, 5 x 10e6 cells were incubated with saturating amounts of the respective antibodies. Data acquisition and analysis was performed on a 2-laser FACSCalibur (BDIS). 1 x 10e6 events were acquired but only CD34+CD38− (in AML) or CD34+CD19− cells (in ALL) were stored (storage gate). Matched isotype controls were analyzed using the same storage gate to exclude amplification of unspecific events. In-between each acquisition, the cytometer was vigorously flushed to prevent carry-over of cells from previous acquisitions. In addition, 10.000 ungated events were acquired to compare the CD34+Lin− stem/progenitor cells with the dominant leukemic cell clone.

In all eight patients with NG2-positve leukemia, cells with a stem cell-like immunophenotype were shown to lack expression of NG2. Instead, flow cytometry analyses revealed a close correlation between expression of the myeloid differentiation marker CD33 and increasing levels of NG2 on maturing AML cells. Similarly, by LightCycler RT-PCR CD34+CD19+ cells showed higher expression of NG2-RNA compared with CD34+CD19− cell in both ALL samples. Our results demonstrate that NG2 is not expressed on primitive leukemic cells but is upregulated with differentiation within the leukemic cell clone. This hampers the use of NG2 expression as a sensitive marker for MRD detection as expression of NG2 does not allow detection of relapse-initiating leukemic stem cells.
Independent Prognostic Significance of Cytogenetics, Immunophenotype, Tumor Burden, and Age in Acute Myeloid Leukemia. Analysis of Patients Treated within PALG 1999 Prospective, Randomized Study.
Authors: Grosicki Sebastian , Holowiecki Jerzy , Giebel Sebastian , Haus Olga , Stella-Holowiecka Beata , Jagoda Krystyna , Bartkowska-Chrobok Aleksandra
Published: April 2020

Acute myeloid leukemia (AML) refers to a group of distinct diseases that differ with regard to their genetic charakteristics, clinical features, response to therapy, and prognosis. Patients require precise initial evaluation of risk factors including cytogenetics and immunophenotype to plan an optimal treatment program.

In this study we analyzed impact of cytogenetics, cell surface marker expression and clinical features on complete remission (CR) rate, leukemia-free survival (LFS) and overall survival (OS).

Four-hundred-forty-five AML patients, aged 18–60 years, treated within a multicentre study by the Polish Adult Leukemia Group (PALG 1999 Study) between 1999–2002 were included in the analysis. Induction therapy consisted of daunorubicine+cytarabine+/−cladribine, consolidation included two courses containing high-dose cytarabine (HAM, HD-AraC+/−cladribine).

In multivariate analysis the following factors were found to be associated with poor outcome:

For CR rate: unfavorable cytogenetics according to CALGB criteria (RR=5,5, p=0,00001), WBC ≥50 x109/L at diagnosis (RR=3,3, p=0,00007), and age ≥50 (RR=1,9, p=0,021). For LFS: unfavorable cytogenetics (RR=1,7, p=0,026), and lack of CD117 expression on leukemic cells (RR=1,8, p=0,026). For OS: unfavorable cytogenetics (RR=1,8, p=0,00002), WBC ≥50 x109/L at diagnosis (RR=1,1, p=0,0007), age ≥50 (RR=1,6, p=0,002), and lack of CD33 expression on leukemic cells (RR=1,7, p=0,014).

CONCLUSIONS: In this study we demonstrated that besides cytogenetics, other factors including particular phenotypic features, as well as initial tumor burden, and age independently influence outcome of AML patients. Results of the analysis provide practical information that could be taken into account when planning individualized treatment including indications for high-dose therapy followed by bone marrow transplantation.