scholarly journals Two-dimensional electrophoresis protein profiles of HL-60 and CCRF-CEM cell lines treated with epigenetic modification drugs

2019 ◽  
pp. 10-23
Author(s):  
Aziee Sudin ◽  
Haiyuni Mohd Yassim ◽  
Shafini Mohamed Yusoff ◽  
Shaharum Shamsuddin ◽  
Ridhwan Abdul Wahab ◽  
...  
Keyword(s):  
Cell Lines ◽  
Dna Methyltransferase ◽  
Epigenetic Modification ◽  
Trichostatin A ◽  
Lymphoblastic Leukemia ◽  
Genetic Alterations ◽  
Epigenetic Modifications ◽  
Hematopoietic Stem ◽  
2D Gel

Leukemia is classified as a malignant disease of hematopoietic stem cells (HSCs) that fails in cell differentiation but preserve their self-renewal. It is caused by genetic alterations and epigenetic modifications resulting in the activation or inactivation of particular genes for transcription. Epigenetic causes changes in gene expression without any alteration in the DNA sequence. The most common epigenetic modifications are DNA methylation and histone acetylation. 5-Azacitidine (5-Aza) is a DNA methytransferase inhibitor (DNMTi) that inhibits DNA methyltransferase enzymes resulting in hypomethylation. Trichostatin A (TSA) is a histone deacetylase inhibitor which inhibits deacetylation of both histone and non-histone proteins resulting in chromatin relaxation. This present study focused on the alteration of proteome profile on 2D gel electrophoresis (2-DE) induced by 5-Aza and TSA in HL-60 and CCRF-CEM cell lines as in vitro model to represent acute promyelocytic leukemia (APL) and T-lymphoblastic leukemia (T-ALL), respectively. Total proteins of untreated and 5-Aza/TSA-treated HL-60 and CCRF-CEM cell lines were extracted using urea/thiourea buffer and stained with Coomassie Blue. Comparative analysis of untreated and 5-Aza/TSA-treated HL-60 and CCRF-CEM was performed by PDQuest software. Qualitative analysis identified 190-659 protein spots detected in untreated, 5-Aza and TSA-treated HL-60 and CCRF-CEM. Quantitative comparison analysis was analyzed by over 2-fold change in 5-Aza/TSA-treated cells compared to untreated. One and eight upregulated proteins were detected in 5-Aza and TSA-treated HL-60, respectively. While five and one upregulated proteins were detected in 5-Aza and TSA-treated CCRF-CEM, respectively. These preliminary results suggested that 5-Aza and TSA induced proteome profiles alterations due to their inhibition effects in HL-60 and CCRF-CEM cell lines.

DNMT3A Mutation Leads to Hematopoietic Dysregulation.

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 2382-2382
Author(s):  
Jie Xu ◽  
Wei-na Zhang ◽  
Tao Zhen ◽  
Yang Li ◽  
Jing-yi Shi ◽  
...  
Keyword(s):  
Transcriptional Activation ◽  
Dna Methyltransferase ◽  
De Novo ◽  
Conflicts Of Interest ◽  
Epigenetic Modification ◽  
Hematopoietic Cells ◽  
In Vitro Assays ◽  
Clinical Samples ◽  
Hematopoietic Stem

Abstract Abstract 2382 Epigenetic modification process is required for the development of hematopoietic cells. DNA methyltransferase DNMT3A, responsible for de novo DNA methylation, was newly reported to have a high frequency of mutations in hematopoietic malignancies. Conditional knock-out of DNMT3A promoted self-renewal activity of murine hematopoietic stem cells (HSCs). However, the role of mutated DNMT3A in hematopoiesis and its regulative mechanism of epigenetic network mostly remain unknown. Here we showed that the Arg882His (R882H) hotspot locus on DNMT3A impaired the normal function of this enzyme and resulted in an abnormal increase of primitive hematopoietic cells. In both controlled in vivo and in vitro assays, we found that the cells transfected by R882H mutant promoted cell proliferation, while decreased the differentiation of myeloid lineage compared to those with wild type. Analysis of bone marrow (BM) cells from mice transduced by R882H reveals an expansion of Lin−Sca-1+C-kit+ populations and a reduction of mature myeloid cells. Meanwhile, a cluster of upregulated genes and downregulated lineage-specific differentiation genes associated with hematopoiesis were discovered in mice BM cells with R882H mutation. We further evaluated the association of mutated DNMT3A and HOXB4 which was previously detected to be highly expressed in clinical samples carrying R882 mutation. Compared with wildtype DNMT3A, R882H mutation disrupted the repression of HOXB4 by largely recruiting tri-methylated histone 3 lysine 4 (H3K4). Taken together, our results showed that R882H mutation disturbed HSC activity through H3K4 tri-methylation, and transcriptional activation of HSC-related genes. Disclosures: No relevant conflicts of interest to declare.

Disruption Of CD9 Expression Affects Adhesion, Migration and Engraftment Of Pre-B Lymphocytes

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 1222-1222
Author(s):  
Marie-Pierre Arnaud ◽  
Audrey Vallée ◽  
Guillaume Robert ◽  
Anne-Gaelle Rio ◽  
Elisabetta Dondi ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Cell Surface ◽  
B Lymphocytes ◽  
Cell Lines ◽  
Actin Polymerization ◽  
Lymphoblastic Leukemia ◽  
Cellular Adhesion ◽  
Hematopoietic Stem

Abstract Introduction CD9 is a membrane protein, member of the tetraspanin family. Recent publications have reported the role of CD9 on engraftment of hematopoietic stem cells, and on cancer stem cell potential. The expression of CD9 has been correlated to the risk of metastases and to a poor clinical outcome in various types of cancer. Surprisingly, CD9 protein is downregulated in ETV6/RUNX1 pre-B acute lymphoblastic leukemia. The purpose of our study is to investigate the effect of CD9 expression on migration and engraftment abilities of pre-B lymphocytes. Materials and Methods The CD9-positive Nalm6 and REH (ETV6/RUNX1) pre-B cells were used. By lentiviral transduction of shRNA targeting mRNA, we generated Nalm6 and REH cell lines depleted in CD9 protein. Engraftment tests were performed in vivo using Nod Scid Gamma immunodeficient mice. REH and Nalm6 cells were detected in bone marrow by CD10 and respectively CD19 or HLA-DR labelling. Ability of the different cell lines to adhere on fibronectin and to migrate through double chambers system in response to CXCL12 were measured in vitro. We also investigated the presence of membrane villosities on REH and REH shCD9 cell surface by scanning electron microscopy. Finally, F-actin polymerization after CXCL12 stimulation was measured by rhodamin-phalloidin labelling. Results In vivo engrafments tests showed that the human cells detected in bone marrow is strongly enriched in CD9 positive cells compared to the initially injected population. This result suggests that CD9 facilitates pre-B lymphobasts homing. An in vitro analysis of adhesion on fibronectin demonstrated that cellular adhesion is dependent on membrane expression of CD9. As well, the more CD9 is expressed, the higher the migration rate in response to CXCL12 chemokine is. The analysis of membrane villosities on REH cell surface revealed that cells over expressing CD9 had longer villosity than shCD9 transducted cell lines. Moreover, F-actin labelling after CXCL12 stimulation showed an increased F-actin polymerization in CD9-positive cells and the formation of actin extensions. Conclusion We provide novel evidence that CD9 is a key player of pre-B lymphoblasts engraftment, adhesion and CXCL12 dependant migration. CD9 expression is related to actin remodelling. We are now investigating a potential link between CD9 and RAC1 activation in response to CXC12. Therefore, the expression level of CD9 could impact leukemic blasts abilities to spread and be responsible of relapses. This work is supported by CNRS, University of Rennes 1, University Hospital of Rennes, la Ligue Régionale contre le Cancer (committee 22, 35 and 56) (MPA, VG, MBT), SFR Biosit UMS 3480 (VG, MBT), Association Laurette Fugain (VG) and Europe Career Integration Grant (MBT). Disclosures: No relevant conflicts of interest to declare.

scholarly journals Germline RUNX1 Variation and Predisposition to Childhood Acute Lymphoblastic Leukemia

2021 ◽  
Author(s):  
Yizhen Li ◽  
Meenakshi Devidas ◽  
Wentao Yang ◽  
Stuart S Winter ◽  
Wenjian Yang ◽  
...  
Keyword(s):  
Acute Lymphoblastic Leukemia ◽  
T Cell ◽  
Lymphoblastic Leukemia ◽  
Ectopic Expression ◽  
Genetic Alterations ◽  
Dominant Negative ◽  
Transcription Activator ◽  
Hematopoietic Stem

RUNX1 is a transcription factor critical for definitive hematopoiesis and genetic alterations in RUNX1 have been implicated in both benign and malignant blood disorders, particularly of the megakaryocyte and myeloid lineages. Somatic RUNX1 mutations are reported in B- and T-cell acute lymphoblastic leukemia (B-ALL and T-ALL), but germline genetic variation of RUNX1 in these lymphoid malignancies have not been comprehensively investigated. Sequencing 4,836 children with B-ALL and 1,354 cases of T-ALL, we identified 31 and 18 unique germline RUNX1 variants in these two ALL subtypes, respectively. RUNX1 variants in B-ALL were predicted to have minimal impact. By contrast, 54.5% of variants in T-ALL result in complete or partial loss of RUNX1 activity as a transcription activator in vitro, with dominant negative effects for 4 variants. Ectopic expression of dominant negative deleterious RUNX1 variants in human CD34+ cells repressed differentiation into erythroid, megakaryocytes, and T cells, while promoting differentiation towards myeloid cells. We then performed chromatin immunoprecipitation profiling in isogenic T-ALL models with variants introduced by genome editing of endogenous RUNX1. We observed highly distinctive patterns of DNA binding and target genomic loci by RUNX1 proteins encoded by the truncating vs missense variants. The p.G365R RUNX1 variant resulted in a novel methylation site in RUNX1 and alteration in its interaction with CBFβ. Further whole genome sequencing showed that JAK3 mutation was the most frequent somatic genomic abnormality in T-ALL with germline RUNX1 variants. Consistently, co-introduction of RUNX1 variant and JAK3 mutation in hematopoietic stem and progenitor cells in mouse gave rise to T-ALL with early T-cell precursor phenotype in vivo, compared to thymic T-ALL seen in mice with JAK3 mutation alone. Taken together, these results indicated that RUNX1 is an important predisposition gene for ALL, especially in T-ALL and also pointed to novel biology of RUNX1-mediated leukemogenesis in the lymphoid lineages.

84 Aging Model in Pig Oocytes as Viewed Through DNA Epigenetic Modification

2021 ◽  
Vol 99 (Supplement_1) ◽  
pp. 108-109
Author(s):  
Haley A Arena ◽  
Kimberly Sprungl ◽  
Skyla Reynolds ◽  
Brian D Whitaker
Keyword(s):  
In Vitro Maturation ◽  
In Vitro Model ◽  
Time Window ◽  
Epigenetic Modification ◽  
Trichostatin A ◽  
Epigenetic Modifications ◽  
Optimal Time ◽  
Vitro Model ◽  
Effects Of Aging

Abstract Oocytes of older animals are less likely to be fertilized during the optimal time window post ovulation, resulting in the potential diminished fertilization and embryonic development success. The activity of the epigenetic modifications during this period is a possible target to reverse these damaging effects of aging. The objective of this study was to study the effects of aging during in vitro oocyte maturation in pigs on epigenetic modifications. Oocytes (n = 54) were matured with or without Trichostatin A (TSA; 100 ng/mL), a known meiotic inhibitor, for 24 h, then for an additional 16 h without TSA or hormones for a total of 40 h. At the end of maturation, oocytes were denuded and their zona pellucida’s removed. Oocytes were stained with anti-5-methylcytosine (5mC, 1:500). Fluorescent images of the oocytes were acquired, images were analyzed using ImageJ, and data analysis was performed using ANOVA and Tukey’s test. Oocytes matured with TSA had significantly greater (P < 0.05) levels of DNA methylation by the end of in vitro maturation compared to those not supplemented with TSA These results suggest that TSA can be used to develop an in vitro model to study the effects of epigenetic modifications in oocytes from aged livestock.

scholarly journals Lomeguatrib Increases the Radiosensitivity of MGMT Unmethylated Human Glioblastoma Multiforme Cell Lines

2021 ◽  
Vol 22 (13) ◽  
pp. 6781
Author(s):  
Anna Kirstein ◽  
Daniela Schilling ◽  
Stephanie E. Combs ◽  
Thomas E. Schmid
Keyword(s):  
Glioblastoma Multiforme ◽  
Cell Lines ◽  
Dna Methyltransferase ◽  
Treatment Options ◽  
Treatment Resistance ◽  
Cell Cycle Distribution ◽  
Human Glioblastoma ◽  
Human Glioblastoma Multiforme ◽  
Mgmt Protein

Background: Treatment resistance of glioblastoma multiforme to chemo- and radiotherapy remains a challenge yet to overcome. In particular, the O6-methylguanine-DNA-methyltransferase (MGMT) promoter unmethylated patients have only little benefit from chemotherapy treatment using temozolomide since MGMT counteracts its therapeutic efficacy. Therefore, new treatment options in radiotherapy need to be developed to inhibit MGMT and increase radiotherapy response. Methods: Lomeguatrib, a highly specific MGMT inhibitor, was used to inactivate MGMT protein in vitro. Radiosensitivity of established human glioblastoma multiforme cell lines in combination with lomeguatrib was investigated using the clonogenic survival assay. Inhibition of MGMT was analyzed using Western Blot. Cell cycle distribution and apoptosis were investigated to determine the effects of lomeguatrib alone as well as in combination with ionizing radiation. Results: Lomeguatrib significantly decreased MGMT protein and reduced radiation-induced G2/M arrest. A radiosensitizing effect of lomeguatrib was observed when administered at 1 µM and increased radioresistance at 20 µM. Conclusion: Low concentrations of lomeguatrib elicit radiosensitization, while high concentrations mediate a radioprotective effect.

scholarly journals 883 An anti-carcinoma monoclonal antibody (mAb) NEO-201 can also target human acute myeloid leukemia (AML) cell lines in vitro

2021 ◽  
Vol 9 (Suppl 3) ◽  
pp. A925-A925
Author(s):  
Alessandra Romano ◽  
Nunziatina Parrinello ◽  
Sara Marino ◽  
Enrico La Spina ◽  
Massimo Fantini ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Functional Analysis ◽  
Epithelial Cells ◽  
Cell Line ◽  
Cell Lines ◽  
Lymphoblastic Leukemia ◽  
Phase 1 ◽  
Adcc Activity ◽  
Phenotypic Analysis

BackgroundNEO-201 is an IgG1 mAb targeting variants of CEACAM5/6 and has demonstrated tumor sensitivity and specificity in epithelial cells. Functional analysis has revealed that NEO-201 can engage innate immune effector mechanisms including ADCC and CDC to directly kill tumor cells expressing its target. A recent Phase 1 clinical trial at the NCI has determined both safety and recommended Phase 2 dosing. We have also seen the expression of the NEO-201 target on hematologic cells, specifically Tregs and neutrophils. Due to epitope being expressed both on malignant epithelial cells as well as several hematologic cells, we designed this study to explore the reactivity of NEO-201 against hematological neoplastic cells in vitro.MethodsPhenotypic analysis was conducted by flow cytometry. Cell lines used were six AML (HL60, U937, MOLM13, AML2, IMS-M2 and OCL-AML3), two multiple myelomas (MM) (OPM2, MM1.S), two acute lymphoblastic leukemia (ALL) (SUP-B15, RPMI8402) and four mantle cell lymphoma (MCL) (Jeko-1, Z138, JVM2 and JVM13). Markers used for flow cytometry analysis were CD15, CD45, CD38, CD138, CD14, CD19 and NEO-201. Functional analysis was performed by evaluating the ability of NEO-201 to mediate ADCC activity against AML cell lines using human NK cells as effector cells.Results5 of 6 AML cell lines tested bind to NEO-201 and the% of positive cells were 47%, 99.5%,100%,100% and 97.8% for HL60, U937, MOLM13, AML3 and IMS-M2, respectively. The% of positive cells in the two MM cell line were 99% and 18% for OPM2 and MM1.S, respectively. NEO-201 binding was not detected in the two ALL and the four MCL cell lines tested. Functional analysis has demonstrated that NEO-201 can mediate ADCC activity against the AML cell line (HL60) tested.ConclusionsThis study demonstrates that NEO-201 mAb’s target is expressed in most of the AML cell lines tested in vitro. In addition, we have shown it can mediate ADCC activity against HL60 cells (AML). Together, these findings provide a rationale for further investigation of the role of NEO-201 in AML as well as MM, further exploring patient PBMCs and bone marrow samples.

Reovirus therapy of lymphoid malignancies

Blood ◽  
2002 ◽  
Vol 100 (12) ◽  
pp. 4146-4153 ◽  
Author(s):  
Tommy Alain ◽  
Kensuke Hirasawa ◽  
Kelly J. Pon ◽  
Sandra G. Nishikawa ◽  
Stefan J. Urbanski ◽  
...  
Keyword(s):  
Progenitor Cells ◽  
Cell Lines ◽  
B Cell Lymphoma ◽  
Lymphoma Cell ◽  
Progeny Virus ◽  
Ras Signaling ◽  
Hematopoietic Stem ◽  
Reovirus Infection ◽  
Lymphoid Malignancies

Reoviruses infect cells that manifest an activated Ras-signaling pathway, and have been shown to effectively destroy many different types of neoplastic cells, including those derived from brain, breast, colon, ovaries, and prostate. In this study, we investigated the reovirus as a potential therapeutic agent against lymphoid malignancies. A total of 9 lymphoid cell lines and 27 primary human lymphoid malignancies, as well as normal lymphocytes and hematopoietic stem/progenitor cells, were tested for susceptibility to reovirus infection. For in vitro studies, the cells were challenged with reovirus (serotype 3 Dearing), and viral infection was assessed by cytopathic effects, viability, viral protein synthesis, and progeny virus production. We present evidence of efficient reovirus infection and cell lysis in the diffuse large B-cell lymphoma cell lines and Burkitt lymphoma cell lines Raji and CA46 but not Daudi, Ramos, or ST486. Moreover, when Raji and Daudi cell lines were grown subcutaneously in severe combined immunodeficient/nonobese diabetic (SCID/NOD) mice and subsequently injected with reovirus intratumorally or intravenously, significant regression was observed in the Raji-induced, but not the Daudi-induced, tumors, which is consistent with the in vitro results. Susceptibility to reovirus infection was also detected in 21 of the 27 primary lymphoid neoplasias tested but not in the normal lymphocytes or hematopoietic stem/progenitor cells. Our results suggest that reovirus may be an effective agent against several types of human lymphoid malignancies.

IPI-504, a Novel, Orally Active HSP90 Inhibitor, Prolongs Survival of Mice with BCR-ABL T315I CML and B-ALL.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 2183-2183 ◽  
Author(s):  
Cong Peng ◽  
Julia Brain ◽  
Yiguo Hu ◽  
Linghong Kong ◽  
David Grayzel ◽  
...  
Keyword(s):  
Cell Lines ◽  
Median Survival ◽  
Combination Treatment ◽  
Lymphoblastic Leukemia ◽  
Hsp90 Inhibitor ◽  
Leukemic Cells ◽  
Prolonged Survival ◽  
Hsp90 Inhibition ◽  
T315i Mutation

Abstract Development of mutations within the kinase domain is a major drug-resistance mechanism for tyrosine kinase inhibitors (TKIs) in cancer therapy. In CML (chronic myeloid leukemia), a disease driven by the constitutively active BCR-ABL oncoprotein, no available TKIs have been effective in treating patients with the BCR-ABL T315I mutation. Heat shock protein 90 (Hsp90) is a highly conserved, constitutively expressed molecular chaperone that facilitates folding of client proteins like BCR-ABL, and affects the stability of these proteins. Several labs have shown that Hsp90 inhibition in vitro results in the degradation of BCR-ABL T315I and induces potent killing of these cell lines. However, these results have not been demonstrated in animal models for BCR-ABL-induced CML and B-ALL (B-cell acute lymphoblastic leukemia, a disease that does not respond well to TKIs including imatinib and dasatinib). Thus, IPI-504, an orally administered Hsp90 inhibitor, was evaluated in murine models of CML and B-ALL. Treatment of mice with wild type (WT)- or T315I BCR-ABL-induced CML with IPI-504 resulted in BCR-ABL protein degradation and a decrease in circulating BCR-ABL positive cells. In response to treatment with vehicle the median survival time of WT and T315I CML mice is approximately 20 days. While the T315I CML mice were resistant to imatinib with a median survival of 21 days, IPI-504 (50 and 100 mg/kg, PO TIW) demonstrated dose-dependent prolonged survival of these mice by 30 and 70 days, respectively (p<0.001 for both doses). Both imatinib and IPI-504 similarly prolonged survival of mice with BCR-ABL-WT-induced CML. In the T315I CML mice prolonged survival of the IPI-504 treated cohort was associated with decreased peripheral blood BCR-ABL positive leukemia cells during treatment, less splenomegaly and improved pulmonary histopathlogy at necropsy. In CML mice receiving mixed BCR-ABL-WT- or T315I-transduced donor bone marrow cells, Hsp90 inhibition more potently suppressed T315I-expressing leukemia clones relative to the WT clones, consistent with in vitro studies where T315I BCR-ABL was more sensitive to IPI-504 induced degradation in cell lines than WT BCR-ABL. Combination treatment with IPI-504 and imatinib was more effective than either treatment alone in prolonging survival of mice bearing both WT and T315I leukemic cells. IPI-504 also significantly prolonged survival of B-ALL mice bearing the T315I mutation (p<0.001). These results provide a rationale for use of an Hsp90 inhibitor as a novel approach to overcoming resistance to TKIs as well as the potential for first line combination treatment in CML patients. The potential for IPI-504 to eliminate mutant kinases via Hsp90 inhibition provides a new therapeutic strategy for treating BCR-ABL-induced CML, ALL as well as other cancers resistant to treatment with TKIs.

Upregulation of CD20 on Human Acute Lymphoblastic Leukemia Cells by IL-4 and CpG Motif Containing Oligonucleotides Increases Susceptibility to Rituximab.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1879-1879
Author(s):  
Bart Nijmeijer ◽  
Marianke L.J. Van Schie ◽  
Roelof Willemze ◽  
J.H. Frederik Falkenburg
Keyword(s):  
Cell Lines ◽  
Lymphoblastic Leukemia ◽  
Surface Expression ◽  
Chimeric Antibody ◽  
Transcriptional Level ◽  
Apparent Lack ◽  
Expression Levels ◽  
Cd20 Expression ◽  
Cpg Motif

Abstract Monoclonal antibodies are emerging modalities in the treatment of hematologic malignancies. Rituximab (RTX), a chimeric antibody that recognizes CD20, shows therapeutic efficacy in non-Hodgkin lymphoma (NHL). Precursor-B ALL (pB-ALL) may also express CD20. However, little is known on the activity of RTX in this disease. We determined CD20 surface expression levels on primary pB-ALL cells by flow cytometry following a standardized protocol in which cells are stained with RTX and secondary antibody. CD20 on primary ALL cells was generally absent or expressed at lower levels than on primary NHL cells (median mean fluorescence intensity (MFI): 35.1, range 5–423 in 17 pB-ALL samples, and median MFI 1082, range 440–1818 in 6 NHL samples). The ALL cells that expressed the highest levels of CD20 were similarly susceptible to RTX mediated complement dependent cytotoxicity (CDC) as NHL cells (median 17.8% lysis, range 9.5–98% and 41% lysis, range 13–72%, respectively). However, in pB-ALL cells, RTX activity was strongly limited by CD20 expression levels as lysis strictly correlated with CD20 surface expression (P=0.89), and CD20 negative pB-ALL cells were not killed. Despite apparent lack of surface expression, quantitative RT-PCR (qPCR) demonstrated that all samples expressed CD20 mRNA. Transcriptional expression levels correlated with surface CD20 expression (P=0.72). Therefore we explored upregulation of CD20 as a strategy to augment activity of RTX in ALL. We previously established an in vitro culturing system that supports long-term proliferation of ALL cells. Using the LeidenALL cell lines that were generated in this system, we evaluated the effect of various agents on expression of CD20. Ten LeidenALL pB-ALL cell lines were cultured in the presence or absence of IL-2, IL-3, IL-5, IL-7, IL-15, a CpG motif containing oligonucleotide (CpG), TNFa or IFNg. Culture of LeidenALL cells in the presence of IL-4 resulted in upregulation of CD20 in 4 out of 10 samples, 24 hours after incubation. Culture of LeidenALL cells in the presence of CpG resulted in upregulation of CD20 in another 4 out of 10 samples, 48 hours after incubation. None of the other cytokines affected expression of CD20. IL-4 and CpG displayed synergistic action as co-incubation of LeidenALL cells with IL-4 and CpG resulted in further increased expression of CD20 in 9 out of the 10 samples after 48 hours of co-incubation. Mean CD20 expression increased from MFI 406 (range 14–2668) to MFI 1572 (range 70–4394). qPCR revealed that CD20 was upregulated on a transcriptional level in all of the 10 cell lines. Upregulation of CD20 augmented RTX-mediated CDC (from 41% lysis, range 8–95% to 78% lysis, range 3–93%). Three CD20-negative LeidenALL cell lines that initially were not susceptible to RTX-mediated CDC were lysed after upregulation. In an in vitro antibody-dependent cellular cytotoxicity (ADCC) assay, using peripheral blood mononuclear cells from healthy donors, rituximab-mediated ADCC was increased in 7 out of the 10 samples (from 18% lysis, range 4–48% to 25% lysis, range 6–51%). In conclusion, these results demonstrate that RTX may possess activity in pB-ALL. Activity of RTX in ALL can be potentiated by modulation of the leukemic cells. These insights may allow successful application of rituximab in ALL.

Glucocorticoid Induced Cell Death Is Mediated by Its Effect on Cellular Metabolism and Is Independent of Caspase Activity.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 3475-3475
Author(s):  
Sandeep Gurbuxani
Keyword(s):  
Cell Death ◽  
Cell Lines ◽  
Lymphoblastic Leukemia ◽  
Lactate Concentration ◽  
Cellular Metabolism ◽  
Mechanisms Of Resistance ◽  
Metabolic State ◽  
Childhood All ◽  
Mitochondrial Mass

Glucocorticoids (GCs) such as prednisone and dexamethasone are a crucial component of acute lymphoblastic leukemia (ALL) therapy protocols. Multiple studies in childhood ALL have demonstrated that resistance to GC mediated cell death in vitro and in vivo is the single most important predictor of treatment outcome in childhood ALL. However, the mechanisms of GC mediated cell death as well as the mechanisms of resistance are poorly understood. The present study was undertaken to better understand the mechanism of GC induced cell death and to delineate possible mechanisms of resistance. In the initial experiments performed, multiple ALL cell lines, when treated with dexamethasone, underwent a reduction in the amount of reactive oxygen species (ROS) followed by cell cycle arrest and finally cell death which was unaffected by the presence of a pan-caspase inhibitor z-VAD-fmk. Since the amount of ROS present in a cell is an indicator of the metabolic state of the cell, specifically the amount of oxidative phosphorylation going on in the mitochondria, additional experiments were performed to directly estimate the mitochondrial mass as well as the metabolic state of the cells treated with GCs. While the mitochondrial mass measured by Mitotracker green labeling of mitochondria in the viable cells remained unchanged in cell lines susceptible to low concentrations (nano or micromolar) of dexamethasone, there was a prominent reduction in mitochondrial mass 36 hours after dexamethasone exposure in MOLT-4 cell line that requires several fold higher (millimolar) concentration of dexamethasone to induce cell death. The reduction in ROS was not accompanied by an increase in glycolysis as determined by the measurement of lactate concentration in the culture supernatants either in the susceptible or the resistant cells. Since one possible mechanism of reduction in ROS is increased scavenging by molecules that are dependent on the presence of NADPH generated during glucose metabolism via the pentose phosphate pathway (PPP), additional experiments were performed to determine if chemical inhibition of this pathway could augment dexamethasone induced cell death in ALL cell lines. Indeed, addition of transandosterone, an inhibitor of G6PD, the rate limiting enzyme of the PPP, resulted in significantly increased dexamethasone toxicity. Based on these experiments it can be concluded that GC induced cell death is mediated by its effect on cellular metabolism. Furthermore, this cell death is caspase independent and likely proceeds via a pathway mechanistically distinct from classical apoptosis. Finally, cells resistant to GC induced cell death have evolved mechanisms to adapt to GC induced changes in cellular metabolism and may maintain energy production via alternative pathways such as the PPP shunt that are independent of mitochondria.

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