bluuebunny/crossref_metadata_2025 huggingface dataset:
Cell-Cycle-Dependent Oscillation of GATA2 Expression in Hematopoietic Cells.
Authors: Koga Shinichiro , Yamaguchi Nobuhiro , Abe Tomoko , Minegishi Masayoshi , Tsuchiya Shigeru , Yamamoto Masayuki , Minegishi Naoko
Published: April 2020

Manipulation of hematopoietic stem cells (HSC) is a key issue in transplantation therapy and regenerative medicine, and methods enhancing proliferation of HSC with keeping pluripotency (self-renewal) are awaited. GATA2 is a transcription factor controlling proliferation with keeping pluripotency of HSC (self-renewal), although simple overexpression experiments of GATA2 do not induce proliferation of HSC. In this study, we showed the cell-cycle dependent oscillation of GATA2 expression, high in S phase but low in G1/S and M phase, in leukemic cells, and also in cord-blood CD34+ cells cultured under stimulation of Notch signals. Using green fluorescent protin-GATA2 fusion protein, we demonstrated the cell-cycle specificity of proteasome- dependent degradation of GATA2, which was inhibited by the treatment with Roscovitine, a selective inhibitor of cyclin- dependent kinase (CDK). Immunoprecipitation- immunoblotting analysis demonstrated phosphorylation of GATA2 at CDK-consensus motifs, S0P+1 and T0P+1, and the interaction of cyclin A, Cdk2, and Cdk4 with GATA2. Furthermore, mutants in CDK-consensus phosphorylation motifs exhibited altered expression profiles of GFP fusion proteins. These results indicate cell-cycle dependent control of GATA2 expression through the phosphorylation and interactions with cyclin-CDK systems, suggesting the possible effect of cell-cycle-specific induction of GATA2 expression on the self-renewal of HSC.
Contribution of STAT3 to Activation of Survivin by Granulocyte-Macrophage Colony-Stimulating Factor in CD34+ Cells.
Authors: Gu Lubing , Chiang Kuang-Yueh , Zhu Ningxi , Findley Harry W. , Zhou Muxiang
Published: April 2020

Granulocyte-macrophage colony-stimulating factor (GM-CSF) has been shown to specifically stimulate proliferation and differentiation of CD34+ hematopoietic progenitor cells. Although STAT3 was thought to be essential for the transduction of GM-CSF-induced cell proliferation, the downstream signaling mediated by STAT3 to support cell proliferation and growth has not been completely understood. Because the inhibitor of apoptosis protein (IAP) survivin is believed to regulate cell proliferation and survival via its anti-apoptotic function, we chose to study the link between STAT3 signaling and survivin expression in CD34+ cells. We constructed plasmids containing the survivin promoter sequence and performed luciferase reporter assay in CD34+ KG-1 cells stimulated with GM-CSF. These experiments showed that GM-CSF stimulated survivin promoter activity. Chromatin immunoprecipitation (CHIP) and electrophoretic mobility shift assay (EMSA) revealed that STAT3 binds to the core survivin promoter containing a STAT response element (SRE) TT(N)5AA at sites −264 to −256. Mutation or deletion of this SRE completely abolished the effect of GM-CSF on survivin promoter activity. Furthermore, specific JAK inhibitor and STAT3 siRNA inhibited GM-CSF-induced survivin promoter activity and survivin expression. Inhibition of survivin by STAT3 siRNA or by withdrawal of GM-CSF in a GM-CSF-dependent CD34+ line TF-1 resulted in decreased cell growth and induction of apoptosis. These results suggest that the anti-apoptotic protein survivin is a transcriptional target of STAT3, and that GM-CSF stimulated-CD34+ cell proliferation is regulated by the JAK/STAT3/survivin signaling pathway.
All-Trans Retinoic Acid Accelerates Migration of MOLT3 to SDF-1 α/CXCL12 through the Phosphatidylinositol 3 Kinase (PI3K)/Akt Pathway.
Authors: Jimi Shiro , Matsumoto Taichi , Suzumiya Junji , Hara Shuji , Takamatsu Yasushi , Tamura Kazuo
Published: April 2020

The chemokine stromal cells derived factor-1 (SDF-1)/CXCL12 stimulates lymphocytes, hematopoietic stem cells (HSC) and hematopoietic progenitor cells (HPC) through the corresponding chemokine receptor CXCR4, which modulates homing, retention, and engraftment into the bone marrow. However, regulatory factors for CXCR4 excluding cytokines are totally unknown. Besides, all-trans retinoic acid (ATRA), a drug inducing cell differentiation and apoptosis, increases motility with unknown mechanisms. In the present study, we investigated the intracellular regulatory mechanisms of SDF-1α-induced migration of ATRA-treated cells. We initially surveyed cells exhibiting strongly membrane-associated CXCR4 (M-CXCR4), and found 3 out of 10 different cell lines (MOLT3, U266B1, and Raji). Of those, MOLT3 was identified as the only one enabled to respond to SDF-1α chemoattraction, accompanied by an increase in actin polymerization. When M-CXCR4 in MOLT3 was neutralized or antagonized by an antibody or the antagonist AMD3100, the migration activity to SDF-1α, analyzed using the Boyden chamber method, was totally abolished. ATRA pretreatment for 3 days induced growth inhibition of MOLT3, but apoptosis and differentiation did not occur. ATRA treatment alone had no effects on either CXCR4 gene expression or its total protein expression, and cell motility also did not increase; however, ATRA treatment dose dependently increased M-CXCR4 expression. When SDF-1α was added to the lower chamber, migration activity of cells pretreated by different doses of ATRA increased dose dependently and reached up to 5 times greater compared with cells lacking ATRA pretreatment. The increased migration activity was blocked by wortmannin, which inhibits phosphatidylinositol 3 kinase (PI3K), AKT inhibitor, and cytochalasin D, which inhibits actin polymerization; U0126, an inhibitor of MEK1/2, did not block the effects of ATRA and SDF-1α. The results indicate that M-CXCR4 is quite important for cell migration, and the intracellular signaling for the M-CXCR4/SDF-1 axis primarily involves the PI3K/Akt pathway, which ATRA positively affected. This is the fist time to show that migration activity to SDF-1 was accelerated by ATRA. Clinical application of this phenomenon may help to augment HSC/HPC homing after transplantation.
PBI-1402: A New Candidate for the Treatment of Anemia.
Authors: Gagnon Lyne , Barabé Jean , Lagraoui Mouna , Grouix Brigitte , Julien Nathalie , Asselin Michel , Gaudreau Dannyck , Sarra-Bournet François , Zacharie Boulos , Penney Christopher , Laurin Pierre , Morin Marie-Josée , Krosl Gorazd , Roy Denis-Claude
Published: April 2020

PBI-1402 is an orally active low molecular weight synthetic compound which is currently in a phase Ib/II clinical trial in patients with anemia associated with cancer and/or chemotherapy. PBI-1402 stimulates the in vitro/ex vivo proliferation and maturation of hematopoietic progenitors (erythroid and myeloid populations) with an activity comparable to EPO. Furthermore, an additive effect is observed in combination with EPO. PBI-1402 enhances the differentiation of pluripotent stem cells: CFU-GEMM, CFU-GM with a predominant effect on BFU-E. Clinical phase I study showed that PBI-1402 is devoid of significant side effects. In addition, the clinical phase I results showed statistical increase (100%, p<0.0001) of relative and absolute reticulocyte count in healthy volunteers after 21 days of oral treatment compared to placebo. Preclinical results demonstrated that PBI-1402 yields an increase in erythrocytes in the systemic circulation. In myeloblated mice that received a syngeneic bone marrow transplant, oral treatment with PBI-1402 resulted in a significant increase in peripheral erythrocyte count, hemoglobin, platelet and bone marrow erythroid progenitor cells. PBI-1402 is targeted as an adjunct to cytotoxic drugs, radiotherapy and bone marrow transplantation for the treatment of anemia.
Mitotic Arrest Deficiency 2 (Mad2) Is Required for Proliferative Synergy of GM-CSF Plus SCF in Hematopoietic Progenitors Via Effects on Apoptosis and Is Mediated by Ligand-Dependent Association of Mad2 with SCF and GM-CSF Receptors.
Authors: Ito Shigeki , Mantel Charlie , Ishida Yoji , Broxmeyer Hal E.
Published: April 2020

Mitotic arrest deficiency 2 (Mad2) is a component of the mitotic spindle checkpoint complex and is essential for accurate chromosome segregation. We previously reported that Mad2 is involved in synergistic proliferation of hematopoietic progenitor cells in response to SCF combined with GM-CSF, but the underlying mechanism remains unclear. Recently, it was reported that Mad2 physically associates with common β chain (βc) of GM-CSF receptor. Because c-Kit constitutively binds to βc, we hypothesized that Mad2 protein physically associates with c-Kit receptor. We examined if Mad2 interacts with c-Kit using human growth factor-dependent cell line, MO7e, which expresses both c-Kit and GM-CSF receptors. Immunoprecipitation assays demonstrated interaction of c-Kit with Mad2. We then examined whether the interaction of Mad2 with c-Kit is cytokine dependent. Mad2 dissociates from c-Kit after stimulation with SCF plus GM-CSF, but not with SCF or GM-CSF alone. Additionally, because bone marrow hematopoietic progenitor cells from Mad2+/− mice lack synergy in response to SCF plus GM-CSF, it is possible that interaction of Mad2 with c-Kit mediates their synergistic proliferative effects. To address this, we examined intracellular cytokine signaling and apoptosis in MO7e cells depleted of Mad2 by RNA interference. We observed no difference between control and Mad2-depleted cells in phosphorylation of Erk1/2 at Thr202/Tyr204 and Akt at Ser473 after synergistic stimulation with SCF plus GM-CSF. In contrast, the percent of apoptosis of Mad2-depleted cells was significantly higher than that of control (26.2±0.7% vs. 20.3±0.4%, P<0.01). This was compatible with the results of apoptosis assays using c-Kit+Lineage− bone marrow cells from Mad2+/− and wild type mice. These results suggest that Mad2 may be involved in synergistic proliferation of hematopoietic progenitor cells via regulation of apoptosis rather than early cytokine signaling. These effects are likely mediated through Mad2 interaction with c-Kit and the beta chain of the GM-CSF receptor.
G2E3 Is Required for Early Embryonic Development.
Authors: Brooks William S. , Kesterson Robert A. , Schoeb Trenton R. , Crawford David F.
Published: April 2020

G2E3 is a putative ubiquitin ligase (E3) identified in a microarray screen for genes encoding mitotic regulatory proteins and DNA damage responsive genes. This protein was predicted to function as an ubiquitin ligase based on the presence of a c-terminal HECT domain, a common E3 catalytic domain. Three n-terminal PHD domains likely function as protein-protein interaction domains. We have demonstrated that G2E3 localizes to the nucleus and nucleolus of HeLa cells with dynamic localization responsive to external stimuli. For example, G2E3 rapidly delocalized from the nucleolus after treatment with DNA damaging agents, a property shared with several other proteins involved in DNA damage responses. The protein is extremely labile as a result of proteasome-mediated degradation, accumulating in nucleoli and aggresomes after proteasome inhibition. In this study, we determined the effect of G2E3 gene disruption. Mice were generated with a germline mutation inactivating the G2E3 gene. Animals with a heterozygous inactivation of the G2E3 gene were phenotypically normal with no overt change in development, growth, longevity, or fertility. In contrast, mice with disruption of both alleles did not survive to birth. We genotyped mice from G2E3+/− intercrosses at several embryonic stages and found that no homozygous mutant animals were identified as early as E8.5. To determine whether G2E3−/− animals survive through the earliest stages of development, blastocysts were isolated and genotyped by nested PCR. At E3.5, G2E3 gene disruption does not appear to affect viability since the numbers of wild-type, heterozygous knockout, and homozygous knockout blastocysts are similar to the expected 1:2:1 ratio. Therefore, G2E3 gene disruption does not appear to impair viability in the earliest stages of development but causes embryonic lethality prior to or at about the time of implantation. We further show by β-galactosidase staining that G2E3 is predominantly expressed within the central nervous system of the developing embryo and in the early stages of limb bud formation. These studies show that G2E3 plays an essential role in early mammalian development. The development of conditional knockout animals may be very valuable in determining the precise function of G2E3 in development and the DNA damage response.
G-CSF Mobilized Human Mesenchymal Stem Cells Are Found in the Peripheral Blood and Have Telomere Limited Growth Potential.
Authors: Lund Troy C. , Baso Lisa , Orchard Paul J.
Published: April 2020

Mesenchymal stem cells (MSC) are multipotent cells found lining the bone marrow cavity. Their primary function is to support the growth and differentiation of hematologic progenitors. MSC have been shown to differentiate into a variety of cell types including: bone, adipocytes, cartilage, neuron-like, and muscle-like cells. There is mounting evidence that these cells can, under the right circumstances, enter the peripheral circulation. However, MSC have not been routinely isolated from peripheral blood. Granulocyte colony stimulating factor (G-CSF) is commonly used to mobilize hematopoietic stem cells from the bone marrow into the peripheral circulation. We show that G-CSF mobilized peripheral blood also contains a small percentage of MSC although lower than that of bone marrow derived MSC (BMMSC): 0.012% vs 0.04%. Isolates were morphologically similar to BMMSC and were successfully expanded and shown to differentiate into osteogenic and adipogenic lineages in the appropriate differentiation conditions. FACS analysis showed that the cells reliably expressed cell surfaces markers commonly found on MSC including CD105, CD29, CD166, and CD13. They were negative for CD14, CD34, CD133, and CD45. Mobilized peripheral blood derived MSC (MPB-MSC) had limited expansion potential when compared with bone marrow isolated MSC. Most cells appeared to cease cell division 20–25 days after isolation. MPB-MSC did not have any detectable telomerase activity (as determined by TRAP assay) and consequently were found to have undergone significant telomere shortening (shown by Southern analysis.) The rarity of this cell in G-CSF-mobilized peripheral blood and the subsequent tremendous pressure to divide in cell culture are likely contributing factors leading to the telomere loss seen in MPB-MSC. This phenomenon probably also accounts for the observed senescence observed in vitro. While we have conclusively shown that MSC can be found in G-CSF mobilized peripheral blood, the use of such cells for transplant or gene therapy may be of limited potential due to the telomere-restricted capability of expansion.
Characteristics of Adipose Tissue-Derived Mesenchymal Stem Cells.
Authors: Jang Jun Ho , Lee Hyun Woo , Eom Young-Woo , Kang Seok Yun , Park Joon Seong , Choi Jin Hyuk , Kim Hugh C.
Published: April 2020

Background: The aim of this study was to examine the differentiation potentials and characteristics of adipose tissue-derived stem cells (ASCs) in other to evaluate for the ASCs to be used as alternative cell sources of mesenchymal stem cells.

Methods: ASCs were isolated from lipo-aspirated adipose tissues by treatment of collagenase A and cultured in a Dulbecco’s Modified Eagle’s Medium (DMEM). To know the characteristics of ASCs, the expression of cell surface antigens was analyzed by flow cytometry, and the proliferation potentials were estimated by colony forming abilities or capacities of population doubling. The differentiation potentials into adipocytes or osteoblasts were confirmed by accumulation of neutral lipid vacuoles stained with Oil-red O and expression of alkaline phosphatases.

Results: When the nucleated cells were isolated by collagenase treatment after lipo-aspiration, the mean cell yield was about 3.1 X 106 or 1.2 X 106 cells per gram of lipo-aspirate (n=8) processed at 3 or 21 hours, respectively. But cells processed at 21 hours were able to form more colonies than those at 3 hours. Flowcytometric analysis showed that Adipose tissue-derived stem cells (ASCs) have a marker expression that is similar to that of bone marrow stromal cells (BMSCs). ASCs expressed CD44, CD73, CD90, and CD105 and were absent for CD14, CD31 and CD45 expression. When primary cells were plated at 50 or 1000 cells/cm2 on 6-well plates, cumulative population doublings were about 50 times until passage 7 or 13 (approximately 130 days), respectively, and ASCs expanded to 1018 cells. ASCs were multipotent, differentiating to the adipocyte and osteoblast lineages. ASCs did not provoke in vitro alloreactivity of incompatable lymphocytes and, moreover, suppressed mixed lymphocyte reaction (MLR) and lymphocyte proliferative response to mitogen.

Conclusion: Our results suggested that ASCs have highly proliferative potentials, multiple differentiation potentials, and immunosuppressive properties like BMSCs. Therefore, ASCs-based reconstructive therapy could employ allogeneic cells and because of their immunosuppressive properties, ASCs could be an alternative source of BMSCs to treat allogeneic conflicts.
Human Platelet-Derived Factors Regulate Mesenchymal Stem Cell Gene Expression.
Authors: Malischnik Christina , Schallmoser Katharina , Rohde Eva , Reinisch Andreas , Guelly Christian , Lanzer Gerhard , Linkesch Werner , Strunk Dirk
Published: April 2020

The use of animal-derived products during human stem cell processing bears the evident risk of xenogeneic prion, virus, or zoonose contamination. Human platelet lysate (HPL) has recently been recognized as a rich source of cytokines and growth factors with the potential to replace fetal bovine serum (FBS) during ex vivo stem cell manipulation. In this study we compared the gene expression profile of human multipotent mesenchymal stromal/stem cells (MSC) during ex vivo expansion for clinical applications under the aegis of either FBS or HPL.

The Applied Biosystems 1700 Expression Array System was used for full genome expression profiling of MSC after a 12–14 day expansion period in a previously optimized low density expansion system. Data have been obtained from biological as well as technical replicates. A starting amount of 40μg total RNA was directly labeled and DIG-labeled cDNA was hybridized to Human Genome Survey Microarray V2.0. Attribution of regulated genes to biological processes and pathways was done using the PANTHER® db analysis software.

We identified more than 300 genes that are differentially regulated upon culture of MSC in HPL compared to FBS. Biological processes specifically activated in HPL culture include mesoderm development, cell cycle control, hematopoiesis and angiogenesis which interestingly correspond to a considerable proportion of the regenerative function of MSC. In contrast, processes related to cell adhesion and adhesion-mediated signaling, cell structure, cell motility and cell communication are significantly upregulated in MSC after FBS in comparison to HPL culture.

Replacing FBS with HPL not only avoids bovine prion, viral and zoonose contamination of MSC for clinical use. The tightly regulated gene expression profiles under the aegis of human growth factors and cytokines provided by HPL may even help to develop new stem cell therapy strategies.
Gene Expression Profiling of Persistent Polyclonal B-Cell Lymphocytosis Using Whole Genome Microarrays.
Authors: Lawrie Charles H. , Troussard Xavier , Mossafa Hossein , Hatton Chris
Published: April 2020

Persistent polyclonal B-cell lymphocytosis (PPBL) is a rare B-cell disorder characterised by elevated serum levels of polyclonal IgM and persistent lymphocytosis of often bi-nucleated B-cells. The molecular basis of this disorder however remains poorly understood. We therefore used whole genome expression microarrays (Affymetrix U133 plus2.0) to compare the gene expression profile of 14 cases of PPBL with normal lymphocyte populations (6 B cell (CD19+) and 3 T cell (CD3+) samples). PPBL patients (10 female, 4 male) had a median age of 43 (range 28–63) and all were smokers with an average lymphocyte count of 5.34 x 107/ml (range 3.7–9) at time of presentation. Eight of these cases had +i(3q) and eight cases exhibited PCC, four cases displayed both chromosomal abnormalities. Normal lymphocyte populations were obtained from two pools of 12 individuals and individual donors.

Microarray data were first RMA pre-processed and then expression levels of patient samples’ genes compared with levels of both normal B cell and T cell samples. ANOVA (P<0.01) was used to identify differentially expressed genes. Thirty-six genes were found to be up-regulated and 157 genes were down-regulated in all patient samples by at least 2-fold.

Components of the AP-1 transcription complex (FOS, FOSB and JUN) were found to be highly up-regulated in all PPBL samples with an average of 7.2, 7.9 and 31.6 fold expression respectively. The cell cycle regulatory molecule CDC27 was also found to be up-regulated in all patient samples (8.8-fold). Down-regulated genes of interest included the B-cell specific transcription factor BACH2 (3-fold), tumor-suppressor SMAD4 (3.6-fold) and apoptotic genes DRAK1 (3-fold) and CAS (5-fold). These data are currently being validated by quantitative RT-PCR.

We propose that over-expression of AP-1 may have significance in PPBL as increased expression of components of AP-1 have previously been linked with lymphoproliferative disorders including Hodgkin’s lymphoma and splenic marginal zone lymphoma. The underlying reason for over-expression of AP-1 in PPBL remains unknown but two characteristic features of this disease could provide clues; firstly AP-1 up-regulation has been demonstrated to be tobacco-induced in animal models and secondly the LMP1 protein of EBV can result in constitutive expression of the AP-1 complex.
FLJ/P1 Is a New Mitochondrial Dehydrogenase Important in Respiration and Apoptosis.
Authors: Coignet Lionel J. , Johnson Timothy , Nganga Alain , Ghoshal Pushpankur , Houde Christiane , Dolce Jean , Ghosh Debashis , Singh Keshav
Published: April 2020

Leukemia is a neoplastic proliferation of cells of hematopoietic origin that arises following somatic mutation in a single hematopoietic stem cell, the progeny of which forms a clone of leukemia cells. Genetic alterations leading to leukemia transformation of a cell are often associated with major alterations of chromosomes that can be detected by studying cells of the leukemic clone in mitosis. One of these alterations is a chromosomal translocation that is often used to identify genes potentially involved in other type of rearrangements such as deletions. Abnormalities of chromosome band 13q14 occur in hematologic malignancies of all lineages and at all stages of differentiation. Unlike other chromosomal translocations, which are usually specific for a given lineage, the chromosomal translocation t(12;13)(p12;q14) has been observed in both B-cell and T-cell precursor acute lymphoblastic leukemia (ALL), in differentiated and undifferentiated acute myeloid leukemia (AML) and in chronic myeloid leukemia at progression to blast crisis (CML-BC). Recently, we have shown the presence of a myeloid- and lymphoid-specific breakpoint cluster regions within chromosome band 13q14 in acute leukemia (Genes Chromosome Cancer 25:222-229,1999). In addition, a new cell line has been established from one of the lymphoid cases, MUTZ5, that carries a single t(12;13) translocation (Leukemia15:1471-1474, 2001). The molecular characterization of this translocation led to the identification of a new gene, FLJ13639, that is disrupted and lost in the MUTZ5 cell line. This gene shares homologies with the large family of short-chain dehydrogenase reductase (SDR). Furthermore, three transcripts and proteins were found to be differentially expressed for this gene, where P1 is potentially the active form of dehydrogenase, while P2 and P3 are lacking the co-activator site. We previously reported that one of the consequences of the loss of FLJ13639 is the over-expression of CD24 that appears to provide leukemia cells with a proliferation and invasiveness advantage, as well as a certain degree of chemoresistance. In addition, data on patient samples indicated a correlation between survival and CD24High/FLJ/P1Low expression profile (Blood 106 (11), Nov 2005). We show that FLJ/P1 is a new mitochondrial protein that is important for the respiration and apoptosis processes. Restoration of the FLJ/P1 function induced CD24 down-regulation, decreased invasive potential as assessed by Matrigel assay and decreased chemoresistance. FLJ/P1 function restoration might represent a new potential therapy, that could, when combined with the assessment of FLJ/P1-CD234 expression profiles, allow the future development of personalized treatment.
Restoration of Rituximab Sensitivity in CD20 Negative B-Cell Lymphoma Cells; Epigenetic Change of CD20 Expression after Treatment of Rituximab.
Authors: Tomita Akihiro , Hiraga Junji , Kiyoi Hitoshi , Kinoshita Tomohiro , Naoe Tomoki
Published: April 2020

CD20 surface antigen is widely expressed on normal B-lineage cells and B-lymphoid malignant cells. Rituximab, which is a chimeric monoclonal antibody specifically recognizing CD20 surface antigen, works as a molecular targeting drug against CD20 positive lymphoid malignancies. Although combination chemotherapy with Rituximab has significantly improved the survival of CD20 positive lymphoma patients, CD20 negative tumor regression and transformation becomes a considerable problem. Recently, we have identified a patient with CD20(−) diffuse large B-cell lymphoma (DLBCL) transformed from CD20(+) follicular lymphoma after chemotherapies including Rituximab. Here we established RRBL1 (Rituximab resistant B-cell lymphoma 1) cells from this patient’s CD20(−) B-cell lymphoma cells, and analyzed the mechanisms of negative CD20 surface antigen expression. CD20 was not expressed as determined by immunoblotting and FACS analysis, and resistance to Rituximab was observed after cell culture analysis with/without Rituximab. Expression of wild type CD20 mRNA was confirmed by RT-PCR, and no genetic mutation in coding sequence or promoter region was observed. Quantitative RT-PCR showed that CD20 mRNA expression level was almost 100 times lower than that of CD20 positive B-cell lymphoma cells obtained from DLBCL patients. These data suggest that lower expression level of CD20 mRNA is closely related to the negative CD20 surface antigen expression, and the aberrant transcription regulation by epigenetic mechanisms can be anticipated. With these data, we thought that CD20 expression could be restored by modulating the transcription regulation. As we expected, CD20 protein re-expression was confirmed by enhancing CD20 mRNA expression after the treatment with specific molecular targeting drugs, and the sensitivity for the Rituximab was significantly restored. Chromatin immunoprecipitation assay suggested that recruitment of co-repressor complexes to CD20 promoter might be related to the regulation of CD20 expression.

Our data may provide us with a new therapeutic strategy, epigenetic therapy or differentiation therapy, combined with molecular targeting therapy using Rituximab, for Rituximab resistant CD20(−) B-cell malignancies.
Frequent Mutation of the PIK3CA Gene in Saudi Arabian Diffuse Large B-Cell Lymphoma.
Authors: Abubaker Jehad A. , Bavi Prashant P. , Al-Harbi Sayer , Atizado Valerie L. , Al-Dayel Fouad , Ezzat Adnan , Uddin Shahab , Al-Kuraya Khawla S.
Published: April 2020

Phosphotidylinositol 3′-kinases are lipid kinases that regulate signaling pathways involved in cell proliferation, adhesion, survival, motility, and are implicated to play an important oncogenic role in many cancer types. The PIK3CA gene encodes the p110α catalytic subunit of PI3K-like gene. Recently, high frequencies of PIK3CA somatic mutations have been detected in various kinds of human cancers notably colorectal, gastric, and breast cancers. It has also been shown that somatic mutations in the PIK3CA gene have been associated with a higher expression of p-AKT in certain cancers. However, the role of PIK3CA mutations in hematological malignancies has not been elucidated in detail. Therefore, we tested whether PIK3CA is subject to mutations in diffuse large B-cell lymphoma (DLBCL). Exons 9 and 20, encoding the highly conserved helical and kinase domains of PIK3CA, which account for 85% of somatic mutation in PIK3CA gene, were subjected to direct sequencing analysis in 219 cases of DLBCL. Somatic missense mutations were observed in 17 of 212 (8%) DLBCL cases. Out of which, 10 mutations (60%) occurred at codon 1047 in the kinase domain, which is one of the previously reported hot spot within the PIK3CA gene. Additionally, a silent polymorphism (T1025T) was found in one of the DLBCL sample. Moreover, activation of AKT was observed in 14 out of 17 (82%) of the DLBCL harboring PIK3CA mutations. Further, 9 out of 17 cases showed high level of pAKT. Additionally, 12 out of 17 DLBCL patients with PIK3CA mutations showed over-expression of the proliferative marker Ki-67. Expression of high level of pAKT was significantly correlated with over-expression of Ki-67 (p=0.0097). Overall survival in DLBCL cases harboring PIK3CA mutation showed poor survival as compared to the DLBCL cases lacking mutations though it was not statistically significant. These data indicate that mutations of PIK3CA play an oncogenic role in DLBCL tumors and argue for a role of PIK3CA targeting in treatment of DLBCL patients.
AML1/ETO-Mediated Lysozyme Repression Is Independently Relieved by Inhibitors of DNA Methylation and Histone Deacetylation.
Authors: Claus Rainer , Fliegauf Manfred , Stock Michael , Duque Jesus , Kolanczyk Mateusz , Lübbert Michael
Published: April 2020

The human lysozyme (LZM) gene, a marker gene for myeloid-specific development, is highly methylated in immature myeloid and in non-myeloid cells (all LZM-negative), and unmethylated in LZM-expressing mature phagocyte cells. Thus this gene provides an excellent model for investigating differentation-associated DNA methylation changes during myelopoiesis. There is now increasing evidence that LZM (containing five perfect consensus binding sites for AML1/RUNX1 in its 5′ region) is repressed by the AML1/ETO chimeric transcription factor (Fliegauf et al, Oncogene 23:9070–81, 2004), and this repression can be relieved by siRNA-mediated AML1/ETO depletion in AML1/ETO-positive Kasumi-1 cells (Dunne et al., Oncogene, 2006). Recently, AML1/ETO has also been implicated in gene-specific epigenetic repression of interleukin-3 (Liu et al, Cancer Res 65, 1277–84, 2005).

By extensive methylation analyses of the LZM gene including bisulfite sequencing, we now demonstrate marked demethylation in both the CpG-poor 5′ region and the exonic CpG island after treatment of Kasumi-1 cells with non-cytotoxic concentrations of the DNA methyltransferase (DNMT) inhibitor 5-aza-2′-deoxycytidine (5-azaCdR), which was not associated with cellular differentiation. By Northern blot analysis, LZM mRNA levels in Kasumi-1 cells but not in AML1/ETO-negative HL-60 and U-937 cell lines were specifically and independently upregulated upon treatment with 5-azaCdR and, to a lesser extent, with the histone deacetylase (HDAC) inhibitor Trichostatin A (TSA). Combined treatment with subliminal concentrations of 5-azaCdR and TSA applied in different schedules did not reveal synergistic effects on LZM transcription. Relative chromatin accessibility of the LZM 5′ region, as detected by “MspI protection” assay, and associated with partial demethylation in several myeloid cell lines, was increased in Kasumi-1 with 5-azaCdR-induced further DNA demethylation, but not by TSA. As shown by chromatin immunoprecitation, TSA increased the acetylation of histones H3 and H4 both in the 5′ flanking region and exonic CpG island. In a U-937 inducible model, antagonization of AML1/ETO-mediated repression of LZM was achieved by TSA, implying that the histone deacetylation in this region of the human LZM gene is mediated by AML1/ETO protein.

In conclusion, we demonstrate functional interactions between DNA methylation and histone modifications in mediating LZM gene repression which implicate AML1/ETO as one component involved in local chromatin remodelling. Interestingly, inhibitors of DNA methylation and histone deacetylation independently relieve repression of this CpG-poor gene in AML1/ETO-positive cells.
Expression of ED-B Fibronectin in Tissues from Human Hematologic Malignancies.
Authors: Sauer Stefanie , Hoffmann Jens , Menrad Andreas , Durkop Horst , Menssen Hans D.
Published: April 2020

Newly formed blood vessels in tissues infiltrated by hematologic malignancies or solid cancers are strikingly different from normal healthy counterparts. Notoriously, they are characterized by vascular shunts, uneven vessel diameters, and endothelial fenestrations. In addition, for the vast majority of solid cancers the extracellular matrix of such vessels was found to contain a type of fibronectin that has an extra-domain B (ED-B), which is expressed during embryonic development and during major tissue remodeling, but not in normal adult organs. The unique expression pattern attributed to ED-B suggests that it may be well suited for vascular targeting. Preliminary data has shown that ED-B expression may also be detected in hematologic malignancies. We have analyzed a large series of human samples of different hematological malignancies, non-malignant or reactive lymphatic conditions, and normal lymphatic and hematopoietic tissues for ED-B expression, using immunohistochemistry (IHC; mAbs BC1, L19, and the newly created MX1) on paraffin sections after steam pressure-based epitope recovery. Consistent with the data from solid tumors, ED-B expression was not detected in normal lymph nodes and lymphatic tissues. However, considerable but variable vessel-associated ED-B expression was detected in the following tissue samples of hematologic malignancies: 56/59 (95%) of Hodgkin lymphomas, 33/36 (91.7%) of DLBCLs, 32/33 (97%) of follicular lymphomas, 17/18 (94.4%) of mantle cell lymphomas, 14/15 (93.3%) of anaplastic large T cell lymphomas, 16/16 (100%) of multiple myelomas,13/14 (92.8%) of B-CLL samples, and in 5/6 (83.3%) samples from chronic myeloproliferative bone marrow, and in 3/3 (100%) from acute leukemia. Reactive lymph nodes (9/10; 90%) showed a faint ED-B expression in the vascular walls, whereas in hypertrophic tonsils (n=5) and healthy or reactive bone marrow (n=12) ED-B expression was rarely detected. Consistent with the literature, ED-B expression was not detected in tissue samples from normal lymph nodes (n=5). Immunofluorescence was used to identify co-localization of ED-B fibronectin and blood vessel-associated epitopes (eg CD34). Also, results will be presented from experiments in which lymphoma and leukemia animal models were treated with a novel IL2 immunocytokine specifically targeted to the ED-B fibronectin (L19-IL2). Preliminary results from these models suggest that hematopoietic malignancies contain ED-B+ vessels. Our study indicates the presence of vessel-associated ED-B in the vast majority of tissue samples from patients with varied hematologic malignancies, suggesting a potential approach for clinical investigation with targeted immunocytokines.
Lack of Increased Gene Promoter Methylation at Relapse in Acute Promyelocytic Leukemia (APL).
Authors: Zhou Da-Cheng , Thompson Reid , Kim Haesook , Greally John , Gallagher Robert
Published: April 2020

Increasing methylation of gene-regulatory DNA sequences has been associated with the progression of malignancies, and, in APL, the pathogenetic PML-RARα fusion gene has been associated with DNA methyltransferase activity, which might contribute to this process. We, therefore, hypothesized that promoter methylation levels would be increased in APL samples at disease relapse. Preliminary studies of a few CpG-enriched gene promoters, using highly quantitative pyrosequencing technology, demonstrated considerable individual-specific variation in DNA methylation. Hence, we used matched pretreatment and relapse bone marrow and/or peripheral blood samples with >85% promyeloblasts after low-density gradient enrichment from 4 APL patients. High quality DNA was prepared from fresh samples by a guanidinium-cesium chloride gradient procedure. Global analysis of CpG-enriched DNA was performed by the HpaII tiny fragment Enrichment by Ligation-mediated PCR (HELP) assay (Khulan, et al, Genome Res. 16, 1046Khulan, et al, Genome Res. 16, 2006), using a microarray representing the promoters of 24,000 human genes. The HELP assay relies upon a comparative genomic hybridization of HpaII (methylation-sensitive) versus MspI (methylation-insensitive) digested genomic DNA and is able to distinguish populations of methylated loci from hypomethylated (HO) loci. Based on the mean centered log ratio (MCLR) of HpaII/MpaI (Khulan, et al), there were negligible global changes in methylation between the 4 matched pretreatment and relapse samples. Using cut-off values to select for hypermethylated (HR) DNA (MCLR ≤0.0) and more completely HO DNA (MCLR ≥1.0) revealed some decrease in the ratio of HR:HO DNA at relapse in 3 of the 4 patients, primarily due to an increased fraction of HO DNA (see table).

We then assessed relapse-associated changes in methylation in 3,908 promoters in which the change from pretreatment to relapse was associated with p <0.05 (paired t-tests) across all 4 data sets. A change from HO to HR was defined as a decrease in the MCLR by ≥2-fold in the relapse group; a change from HR to HO was the opposite, i.e., an increase in MCLR by ≥2-fold. There was a change from pretreatment HO to relapse HR for 563 loci (7.5% of effective HO promoters) and from pretreatment HR to relapse HO for 949 promoters (8.6% of effective promoters). More stringently, when promoters were selected for a change in MCLR from pretreatment HO ≥1.0 to relapse HR ≤0.0 or, conversely from pretreatment HR ≤0.0 to relapse HO ≥1.0, 40 and 82 promoters, respectively, were identified with p ≤0.05. These results indicate that changes in gene promoter methylation from pretreatment HR to relapse HO are more common than the reverse, and they are contrary to our pre-study hypothesis that increased global DNA methylation might be a major contributor to molecular progression processes in APL cells leading to clinical relapse.
Different Activation of WNT Signaling Pathway in B-Cell and T-Cell Acute Leukemias.
Authors: Sayitoglu Muge , Hatirnaz Ozden , Erbilgin Yucel , Atalar Fatmahan , Ozbek Ugur
Published: April 2020

WNT signaling pathway proteins function as hematopoietic growth factors and regulate proliferation in normal T-cell and B-cell development. Recent experimental evidence demonstrated that oncogenic transformation in leukemias of both lymphoid and myeloid lineages is dependent on WNT signaling. Not much is known about activation of WNT signaling pathway, its ligands and receptors in hematopoiesis and leukemia pathogenesis. To define its role in leukemia, we aimed to determine mRNA levels of the critical members of WNT pathway (WNT5A, WNT10B, FZ5, β catenin, APC, TCF-1 and LEF-1) by using quantitative real time PCR in acute lymphoblastic leukemia (ALL) patients (T-cell n=42, B-cell n=46 and pre B-cell n=30) and normal hematopoietic cells (bone marrow n=6, peripheral blood n=10, and CD19+ cells from peripheral blood). These genes expressed varying levels in B-cells, preB-cells and T-cells. In the B-cell leukemia patients, WNT5A was expressed notably (OR=58.05 CI 95% 1.63–1219.55, p>0,001). WNT5A directs Ca++ dependent signaling by PKC and a G protein dependent manner which is an alternative pathway for beta-catenin mediated signaling. Also LEF-1 levels were higher in B-ALL patients and APC expression was down regulated when compared to normal tissue (OR=18.81 CI 95% 0.34–5703, p>0.001 and OR=0.212 CI 95% 0.006–8.816, p=0.001, respectively). It is known that LEF-1 blocks APC mediated β catenin nuclear export and activates transcription of various transforming genes, including cyclin, D1, c-myc, MMP7, and LEF-1 itself. WNT5A or WNT10B proteins were not found to be up regulated in preB-ALL whereas APC and LEF-1 gene expressions were increased compared to normal hematopoietic cells (OR=32.97 CI 95% 0.27–1281, 38 p>0.001 and OR=5.57 CI 95% 0.28–89.51, p=0.01, respectively). We found increased TCF-1 expression (7.4 fold) without any β catenin accumulation in T-ALL patients. It is known that TCF-1 in absence of β catenin functions as a tumor suppressor gene. WNT5A, APC and LEF-1 gene expression levels were also different between T-cell, B-cell and preB cell ALL cases. WNT5A expression had the highest levels in B-ALL compared to T-ALL cases, whereas the highest APC expression levels were observed in preB and T-ALL patients. Also LEF-1 expression levels were significantly different between preB and T-cell ALL patients. Taken together these results indicate that WNT signaling genes have abnormal expression and are active in acute lymphoblastic leukemia. This data suggests different WNT activation mechanisms exist in the leukemic transformation in different hematopoietic cells.
Targeting of Heat Shock Protein 32 (Hsp32) in Neoplastic Cells by Styrene Maleic Acid Zinc Protoporphyrin (SMA-ZnPP) Is Associated with Reduced Growth and Induction of Apoptosis.
Authors: Gleixner Karoline V. , Matthias Mayerhofer , Vales Anja , Gruze Alexander , Kneidinger Michael , Hoermann Gregor , Krauth Maria T. , Kondo Rudin , Sonneck Karoline , Samorapoompichit Puchit , Lackner Eva , Greish Khaled , Pickl Winfried F. , Sillaber Christian , Zielinski Christoph C. , Maeda Hiroshi , Valent Peter
Published: April 2020

Heat shock protein 32 (Hsp32), also known as heme oxygenase-1 (HO-1), is a stress-related survival factor that has recently been implicated in enhanced survival of neoplastic cells. We here show that Hsp32/HO-1 is expressed abundantly in primary neoplastic cells in various solid tumors and hematopoietic neoplasms such as acute myeloid leukemia (AML) or chronic myeloid leukemia (CML), and in respective cell lines including the AML cell lines HL60, KG1, KG1a, and U937, CML cell lines K562 and KU812, eosinophilic leukemia cell line EOL-1, mast cell leukemia cell line HMC-1, myeloma cell lines RPMI8226 and U266, breast cancer cell line MDA MB 231, lung cancer cell line A549, pancreatic carcinoma cell line BxPC-3, colon carcinoma cell lines COLO201, COLO205, COLO320DM, and DLD-1, and the ovarian carcinoma cell line OVCAR-3. Expression of Hsp32 mRNA was demonstrable by RT-PCR and Northern blotting, and expression of the Hsp32 protein by Western blotting and immunocytochemistry. The CML-specific oncoprotein BCR/ABL and the transforming oncoprotein KIT D816V that is expressed in neoplastic mast cells, were found to promote expression of Hsp32 in Ba/F3 cells. In order to examine the functional role of Hsp32 in neoplastic cells, a specific siRNA was employed. Expression of Hsp32 siRNA resulted in reduced viability and induction of apoptosis in all cell lines tested. To further explore the value of Hsp32 as a target in neoplastic cells, a novel specific Hsp32-targeting compound, styrene maleic acid copolymer zinc protoporphyrin micelles (SMA-ZnPP) was applied. Exposure to SMA-ZnPP resulted in a significant decrease in proliferation determined by 3H-thymidine uptake, in all cell lines as well as in all primary neoplastic cells tested (AML, n=5; CML, n=5; mastocytosis, n=3; breast cancer, n=2; lung cancer, n=1). As assessed by AnnexinV-staining, Tunel assay and electron microscopy, the growth-inhibitory effects of SMA-ZnPP were found to be associated with induction of apoptosis. In each cell type, the effect of SMA-ZnPP on growth was dose-dependent and found to occur at pharmacologic concentrations (IC50 1–30 μM). Moreover, SMA-ZnPP was found to synergize with various anti-neoplastic drugs (tumor cell lines: cisplatin, leukemias: cytarabine, myeloma: bortezomib) in producing growth inhibition. In summary, these data show that Hsp32/HO-1 is an important survival factor and novel interesting target in various hematopoietic and non-hematopoietic neoplasms. Based on these data, it seems desirable to explore the value of the Hsp32-targeting drug SMA-ZnPP in clinical trials in patients with leukemias and solid tumors.
Mutation in Genes Impacting on the RAS-RAF-MEK-ERK Pathway Are Found at High Incidence in Childhood Acute Lymphoblastic Leukemia at Both Diagnosis and at Relapse.
Authors: Irving Julie , Minto Lynne , Matheson Elizabeth , Case Marian , Bailey Simon , Bown Nick , Hassan Rosline , Harrison Christine , Hall Andrew
Published: April 2020

Deregulation of the RAS-RAF-MEK-ERK signalling cascade, often caused by activating mutations in the RAS family of G-proteins, is a recurrent event in haematological malignancies including childhood acute lymphoblastic leukemia (ALL). However, deregulation of this pathway can also arise from mutational events in genes encoding other proteins which impact on this pathway including FLT3, PTPN11 and BRAF and such mutations have recently been identified in childhood ALL. The combined incidence of mutations in these genes, along with the RAS family is not known, but may be significant.

A comprehensive mutational screen of key exons of NRAS, KRAS2, FLT3, PTPN11 and BRAF was performed in 86 diagnostic and 49 relapse childhood ALL samples using denaturing high performance liquid chromatography (DHPLC) and direct sequencing and/or PCR product cloning. Using wild type cells spiked with cell lines bearing known mutations, the sensitivity of this screening assay was shown to be between 12–25%.

At diagnosis the combined mutation incidence was 35% (30 from 86) made up of NRAS (n=16), KRAS2 (n=15), FLT3 (n=3), PTPN11 (n=2), with 4 patients having more than one mutation. At relapse, 25 % (12 from 49) of cases showed mutations and included NRAS (n=4), KRAS2 (n=6), FLT3 (n=1) and PTPN11 (n=1). No mutations were identified in BRAF. While at relapse the mutated blasts were always the predominant population, in 7 of the 30 diagnostic cases, only a minor subpopulation of blasts housed the mutation and included all of the FLT3 insertion/deletion mutants. In 2 relapsed cases with mutations in KRAS2, mutations were not detectable by DHPLC in the matched diagnostic samples but were apparent at low level when a more sensitive allele-specific PCR was employed (assay sensitivity between 0.1–1%).

These data reveal a significant role of RAS-RAF-MEK-ERK pathway deregulation in leukemogenesis and implicate the pathway in some patients with progression to relapse. In addition, they highlight the genetic heterogeneity of leukemic blasts at diagnosis compared to those at relapse suggesting that clonal selection occurs in response to drug exposure. Inhibitors of the pathway, which are currently undergoing clinical trial, may be a novel therapeutic option for a significant proportion of children with relapsed ALL.