Targeted Therapy of AML1/ETO-Positive AML Cells in Experimental Model.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 3358-3358
Author(s):  
Michal Zapotocky ◽  
Julia Starkova ◽  
Ester Mejstrikova ◽  
Karel Smetana ◽  
Jan Trka
Keyword(s):  
Cell Cycle ◽  
Flow Cytometry ◽  
Cell Line ◽  
Target Genes ◽  
Myeloid Cells ◽  
Surface Markers ◽  
Qrt Pcr ◽  
Hla Dr ◽  
Genes Expression ◽  
Leukaemic Cells

Abstract In t(8;21) acute myeloid leukaemia (AML), the leukaemogenesis is supposed to be promoted by interference with expression of AML1 target genes. Repressor complex associated with AML1/ETO fusion protein recruits class I histone deacetylases (HDAC). Valproic acid (VPA) was found to have an extensive effect on AML blasts, via inhibition of class I HDAC. It was shown previously that VPA treatment disrupts the AML1/ETO-HDAC1 complex from AML1 promoter thus leading to apoptosis at different cell lines. However, there is still lack of in-depth morphological and immunophenotypical proof of the hypothesized restoration of differentiation after treatment with VPA. Although very little is known about AML1 target genes, it was shown that AML1 protein binds to promotor region of IL-3 and PU.1 and regulates their expression. There are also reports demonstrating possible candidate target genes of AML1 transcription factor (BPI, IGFBP7). We aimed to characterize the differentiation effect of VPA on AML1/ ETO-positive leukaemic cells and to determine the expression pattern of selected genes. Kasumi-1 (AML1/ETO-positive) cell line and MV4-11 (MLL/AF4-positive) cells were treated with VPA (0,5 mM and 1,0 mM concentrations) and examined by flow cytometry, morphological evaluation and qRT-PCR. Paediatric patients’ bone marrow samples (AML1/ ETO-positive) from the time of diagnosis were taken for in vitro experiment. We optimised the method of patients’ samples cultivation using conditioned medium with cytokines (IL6, FLT3 ligand, TPO, SCF) and we treated leukaemic cells with VPA. We examined immunophenotype and cell cycle of these samples after 24 and 48 hours of cultivation. We show that treatment of AML1/ETO-positive myeloid cells with HDACi VPA resulted in decreased expression of early myeloid progenitor antigens (CD33/34/117) and increased expression of antigens typical for differentiated myeloid cells (CD11a/11b). Cell morphology, nucleolar morphology and cytochemistry evaluation indicated the maturation process and decreased proliferation activity. All these phenomenons were not observed in control MLL/AF4-positive myeloid cells. We quantified the level of expression of selected genes (PU.1, C/EBPalpha, BPI, IGFBP7) and we observed the increase of genes expression after VPA treatment in AML1/ETO-positive cells. In VPA treated AML1/ETO-positive cells PU.1 increased expression 6.2 times (p<0.001), C/EBPalpha 3 times (p<0.001), BPI 2.6 times (p<0.001) and IGFBP7 7 times (p<0.001). Different situation occurred in MLL/AF4-positive cell line, where PU.1 conversely decreased expression 2.5 times (p=0.01), IGFBP7 decreased 2.4 times (p=0.01) and the expression of C/EBPalpha and BPI remained unchanged (p=0.32 and p=0.75) after VPA treatement. Samples recovered from patientś bone marrow reacted differently to VPA therapy. The first patient increased the expression of HLA-DR (p=0.04) and CD11b (p=0.02) and at the same time decreased the expression of CD38 (p=0.006) and CD117 (p=0.03) surface markers, which represents the signs of differentiation. The second patient’s sample presented with expression of HLA-DR (p=0.1) unchanged; expression of CD11b (p=0.05), CD38 (p=0.002), CD117 (p=0.04) and CD34 (p=0.02) decreased. The concentration of VPA used in our experiment seems to have strong cytotoxic effect on the other patient’s leukaemic cells, as they passed into the apoptosis, the amount of 10% of persistent blast cells was not sufficient for the analysis. Cell cycle examination confirmed the results of the experiment with cell lines; patientś samples treated with VPA decreased the proliferation and the number of cells undergoing apoptosis increased. Taken together, we provide a valid evidence of differentiation of AML1/ETO-positive cell line, demonstrated by flow cytometry and confirmed morphologically. This process goes hand in hand with the increase of the repressed genes expression as measured by qRT-PCR in contrast with non-CBF leukemic cells. Patients’ data are not completely in line with those experimental findings, as the flow cytometry analysis showed uneven changes in surface markers expression pattern. VPA induces differentiation and apoptosis; therefore it seems to be a promising drug in treatment of AML/ETO-positive paediatric AML. Supported by Grant Agency of Charles University 71/2006.

scholarly journals Proposed megakaryocytic regulon of p53: the genes engaged to control cell cycle and apoptosis during megakaryocytic differentiation

2012 ◽  
Vol 44 (12) ◽  
pp. 638-650 ◽  
Author(s):  
Pani A. Apostolidis ◽  
Stephan Lindsey ◽  
William M. Miller ◽  
Eleftherios T. Papoutsakis
Keyword(s):  
Cell Cycle ◽  
Dna Synthesis ◽  
Cell Line ◽  
Target Genes ◽  
Control Cell ◽  
P53 Expression ◽  
Megakaryocytic Differentiation ◽  
Knock Down ◽  
Cell Cycling ◽  
And Control

During endomitosis, megakaryocytes undergo several rounds of DNA synthesis without division leading to polyploidization. In primary megakaryocytes and in the megakaryocytic cell line CHRF, loss or knock-down of p53 enhances cell cycling and inhibits apoptosis, leading to increased polyploidization. To support the hypothesis that p53 suppresses megakaryocytic polyploidization, we show that stable expression of wild-type p53 in K562 cells (a p53-null cell line) attenuates the cells' ability to undergo polyploidization during megakaryocytic differentiation due to diminished DNA synthesis and greater apoptosis. This suggested that p53's effects during megakaryopoiesis are mediated through cell cycle- and apoptosis-related target genes, possibly by arresting DNA synthesis and promoting apoptosis. To identify candidate genes through which p53 mediates these effects, gene expression was compared between p53 knock-down (p53-KD) and control CHRF cells induced to undergo terminal megakaryocytic differentiation using microarray analysis. Among substantially downregulated p53 targets in p53-KD megakaryocytes were cell cycle regulators CDKN1A (p21) and PLK2, proapoptotic FAS, TNFRSF10B, CASP8, NOTCH1, TP53INP1, TP53I3, DRAM1, ZMAT3 and PHLDA3, DNA-damage-related RRM2B and SESN1, and actin component ACTA2, while antiapoptotic CKS1B, BCL2, GTSE1, and p53 family member TP63 were upregulated in p53-KD cells. Additionally, a number of cell cycle-related, proapoptotic, and cytoskeleton-related genes with known functions in megakaryocytes but not known to carry p53-responsive elements were differentially expressed between p53-KD and control CHRF cells. Our data support a model whereby p53 expression during megakaryopoiesis serves to control polyploidization and the transition from endomitosis to apoptosis by impeding cell cycling and promoting apoptosis. Furthermore, we identify a putative p53 regulon that is proposed to orchestrate these effects.

scholarly journals αO-Conotoxin GeXIVA Inhibits the Growth of Breast Cancer Cells via Interaction with α9 Nicotine Acetylcholine Receptors

Marine Drugs ◽  
2020 ◽  
Vol 18 (4) ◽  
pp. 195 ◽  
Author(s):  
Zhihua Sun ◽  
Jiaolin Bao ◽  
Manqi Zhangsun ◽  
Shuai Dong ◽  
Dongting Zhangsun ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Cell Cycle ◽  
Flow Cytometry ◽  
Cell Cycle Arrest ◽  
Cancer Cells ◽  
Cell Line ◽  
Cancer Cell ◽  
Breast Cancer Cell ◽  
Breast Cancer Cells ◽  
Cycle Arrest

The α9-containing nicotinic acetylcholine receptor (nAChR) is increasingly emerging as a new tumor target owing to its high expression specificity in breast cancer. αO-Conotoxin GeXIVA is a potent antagonist of α9α10 nAChR. Nevertheless, the anti-tumor effect of GeXIVA on breast cancer cells remains unclear. Cell Counting Kit-8 assay was used to study the cell viability of breast cancer MDA-MD-157 cells and human normal breast epithelial cells, which were exposed to different doses of GeXIVA. Flow cytometry was adopted to detect the cell cycle arrest and apoptosis of GeXIVA in breast cancer cells. Migration ability was analyzed by wound healing assay. Western blot (WB), quantitative real-time PCR (QRT-PCR) and flow cytometry were used to determine expression of α9-nAChR. Stable MDA-MB-157 breast cancer cell line, with the α9-nAChR subunit knocked out (KO), was established using the CRISPR/Cas9 technique. GeXIVA was able to significantly inhibit the proliferation and promote apoptosis of breast cancer MDA-MB-157 cells. Furthermore, the proliferation of breast cancer MDA-MB-157 cells was inhibited by GeXIVA, which caused cell cycle arrest through downregulating α9-nAChR. GeXIVA could suppress MDA-MB-157 cell migration as well. This demonstrates that GeXIVA induced a downregulation of α9-nAChR expression, and the growth of MDA-MB-157 α9-nAChR KO cell line was inhibited as well, due to α9-nAChR deletion. GeXIVA inhibits the growth of breast cancer cell MDA-MB-157 cells in vitro and may occur in a mechanism abolishing α9-nAChR.

GA Binding Protein (GABP) Is Required for Development and Maturation of Myeloid Cells.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 776-776
Author(s):  
Zhongfa Yang ◽  
Alan G. Rosmarin
Keyword(s):  
Cell Cycle ◽  
Transcription Factor ◽  
Target Genes ◽  
Embryonic Stem ◽  
Myeloid Cells ◽  
Transactivation Domain ◽  
Wild Type ◽  
Floxed Allele

Abstract GABP is an ets transcription factor that regulates transcription of key myeloid genes, including CD18 (beta2 leukocyte integrin), neutrophil elastase, lysozyme, and other key mediators of the inflammatory response; it is also known to regulate important cell cycle control genes. GABP consists of two distinct and unrelated proteins that, together, form a functional transcription factor complex. GABPalpha (GABPa) is an ets protein that binds to DNA; it forms a tetrameric complex by recruiting its partner, GABPbeta (GABPb), which contains the transactivation domain. GABPa is a single copy gene in both the human and murine genomes and it is the only protein that can recruit GABPb to DNA. We cloned GABPa from a murine genomic BAC library and prepared a targeting vector in which exon 9 (which encodes the GABPa ets domain) was flanked by loxP (floxed) recombination sites. The targeting construct was electroporated into embryonic stem cells, homologous recombinants were implanted into pseudopregnant mice, heterozygous floxed GABPa mice were identified, and intercrossing yielded expected Mendelian ratios of wild type, heterozygous, and homozygous floxed GABPa mice. Breeding of heterozygous floxed GABPa mice to CMV-Cre mice (which express Cre recombinase in all tissues) yielded expected numbers of hemizygous mice (only one intact GABPa allele), but no nullizygous (GABPa−/−) mice among 64 pups; we conclude that homozygous deletion of GABPa causes an embryonic lethal defect. To determine the effect of GABPa deletion on myeloid cell development, we bred heterozygous and homozygous floxed mice to LysMCre mice, which express Cre only in myeloid cells. These mice had a normal complement of myeloid cells but, unexpectedly, PCR indicated that their Gr1+ myeloid cells retained an intact (undeleted) floxed GABPa allele. We detected similar numbers of in vitro myeloid colonies from bone marrow of wild type, heterozygous floxed, and homozygous floxed progeny of LysMCre matings. However, PCR of twenty individual in vitro colonies from homozygous floxed mice indicated that they all retained an intact floxed allele. Breeding of floxed GABPa/LysMCre mice with hemizygous mice indicated that retention of a floxed allele was not due to incomplete deletion by LysMCre; rather, it appears that only myeloid cells that retain an intact GABPa allele can survive to mature in vitro or in vivo. We prepared murine embryonic fibroblasts from homozygous floxed mice and efficiently deleted GABPa in vitro. We found striking abnormalities in proliferation and G1/S phase arrest. We used quantitative RT-PCR to identify mechanisms that account for the altered growth of GABPa null cells. We found dramatically reduced expression of known GABP target genes that regulate DNA synthesis and cell cycle that appear to account for the proliferative defect. We conclude that GABPa is required for growth and maturation of myeloid cells and we identified downstream targets that may account for their failure to proliferate and mature in vitro and in vivo.

TET2 Downregulation Promotes AML Cell Proliferation Via Pim-1 Expression

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 5085-5085
Author(s):  
Qingxiao Chen ◽  
Jingsong He ◽  
Xing Guo ◽  
Jing Chen ◽  
Xuanru Lin ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Flow Cytometry ◽  
Cell Proliferation ◽  
Western Blot ◽  
Cell Lines ◽  
Target Genes ◽  
Leukemia Cell ◽  
Leukemic Cells ◽  
Rna Seq ◽  
Knock Down

Abstract Background: Acute myeloid leukemia (AML) is the most common form of acute leukemia in adults which is still incurable although novel drugs and new combination of chemotherapies are used . With the development of genetic and molecular biology technologies, more and more genes are found to be related to leukemogenesis and drug resistance of AML. TET2, a member of the ten-eleven-translocation gene family which can modify DNA by catalyzing the conversion of 5-mehtyl-cytosine to 5-hydroxymethyl-cytosine , is often inactivated through mutation or deletion in myeloid malignancies. Recent research reported that TET2 knock-down can promote proliferation of hematopoietic stem cells and leukemic cells. Also, several clinical studies showed that patients with TET2 mutation or low levels of TET2 expression have more aggressive disease courses than those with normal levels of TET2. However, the mechanism of the phenomenon is unknown. Our aim is to uncover how TET2 protein level is negatively correlated with AML cell proliferation and to provide a better view of target therapy in AML. Methods: We determined the expression levels of TET2 and other target genes in acute leukemia cell lines, bone marrow AML specimens, and peripheral blood mononuclear cells from healthy donors by qRT-PCR and Western blot. We also determined the mutation status of TET2 in AML cell lines. CCK8 and flow cytometry were used to determine cell proliferation, cell apoptosis, and cell cycle profile. Methylation-specific PCR were used to examine the methylation status in gene promoter regions. Also, we developed TET2 knock-down lentivirus to transfect AML cell lines to examine the effect of TET2 depletion. Last, RNA-seq was used to compare gene expression level changes between TET2 knock-down cell lines and the control cell lines. Results: AML cells from AML cell lines (KG-1,U937, Kasumi, HL-60, THP-1, and MV4-11) and AML patients' specimens expressed lower levels of TET2 than those of PBMC from the healthy donor (P<0.05). Among AML cell lines, U937 barely expressed TET2, while KG-1 expressed TET2 at a relatively higher level than those of other AML cell lines. We constructed a TET2 shRNA to transfect KG-1,THP-1,MV-4-11,Kasumi,and HL-60, and used qRT-PCR and western blot to verify the knock-down efficiency. CCK8 confirmed that knocking down TET2 could increase leukemia cell proliferation (P<0.05). Flow cytometry showed that cell cycle profile was altered in TET2 knock-down cells compared to the negative control cells. In order to identify target genes, we performed RNA-seq on wildtype and TET2 knockdown KG-1 cells and found that the expression of cell cycle related genes, DNA replication related genes, and some oncogenes were changed. We focused on Pim-1, an oncogene related to leukemogenesis, which was significantly up-regulated in the RNA-seq profile. Western blot and qPCR verified the RNA-seq results of Pim-1 expression in the transfected cells . Also, AML patients' bone marrow samples (n=35) were tested by qPCR and 28 of them were found to express low TET2 but high Pim-1 with the other 7 being opposite. For detailed exploration in expression regulation of Pim-1 via TET2, we screened genes affecting Pim-1 expression and found SHP-1, a tumor suppress gene which is often silenced by promoter methylation in AML. Western blot band of SHP-1 was attenuated in TET2 knockdown KG-1 cells. Moreover, methylation-specific PCR showed that after knocking down TET2 in KG-1 cell line, the promoter regions were methylated much more than the control cells. These results indicated that the function of TET2 in epigenetic modulation plays an important role in regulating Pim-1 expression. Finally, using flow cytometry and CCK8 we surprisingly found that knocking down TET2 expression could lead leukemic cells (KG-1, THP-1 and MV-4-11) more sensitive to Pim-1 inhibitor (SGI-1776 free base) and decitabine (a demethylation agent treating MDS and AML) (P<0.05). Conclusion: Our study showed that knocking down TET2 promoted leukemic cell proliferation. This phenomenon may correlate to Pim-1 up-regulation. Our clinical data also showed that the expression of TET2 and Pim-1 have an inverse relationship. The mechanism of TET2 regulating Pim-1 expression may be related to the epigenetic modulation function of TET2. Finally, we found TET2 downregulation could increase leukemia vulnerability to Pim-1 inhibitor and decitbine, and provide a novel view of target therapy in AML. Disclosures No relevant conflicts of interest to declare.

scholarly journals Evaluation of Anticancer Activity of Olmesartan and Ramipril On A549 Cell Line

2018 ◽  
Vol 11 (3) ◽  
pp. 1351-1357 ◽  
Author(s):  
E Gayathri ◽  
K. Punnagai ◽  
D. Darling Chellathai
Keyword(s):  
Cell Cycle ◽  
Flow Cytometry ◽  
Anticancer Activity ◽  
Cell Line ◽  
A549 Cell ◽  
Enzyme Inhibitor ◽  
A549 Cell Line ◽  
Ic50 Values

Angiotensin Converting Enzyme Inhibitor (ACEI) and Angiotensin II type 1 receptor antagonist (ARBs) are the most efficient cardiovascular drugs and exhibited efficient cytostatic activity in vitro in many malignant and normal cells1.OBJECTIVE: This study aims to assess the anticancer activity of these two drugs in a dose dependant manner using A549 cell line through MTT assay and Cell cycle analysis.. MATERIALS AND METHODS: Ramipril and Olmesartan were added to A549 at various concentrations ranging from 10⁻⁶ to 10mM.The dot plot of the cytotoxicity results were used to extrapolate the IC50 values. The dot plot of flow cytometry results were used to extrapolate the DNA percentage in phases of cell cycle. The plates were read at 570 nm by using a PERCLIN ELMER (multimode reader). Measurements for concentration required for 50% inhibition was noted. RESULTS: Ramipril and Olmesartan were added to A549 at various concentrations ranging from 10⁻⁶ to 10mM.The dot plot of the cytotoxicity results were used to extrapolate the IC50 values. The dot plot of flow cytometry results were used to extrapolate the DNA percentage in phases of cell cycle.

scholarly journals miR-433 Inhibits Neuronal Growth and Promotes Autophagy in Mouse Hippocampal HT-22 Cell Line

2020 ◽  
Vol 11 ◽  
Author(s):  
Chunli Xu ◽  
Qingke Bai ◽  
Chen Wang ◽  
Qiuyu Meng ◽  
Yuming Gu ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Cell Proliferation ◽  
Rna Interference ◽  
Neurodegenerative Diseases ◽  
Cell Line ◽  
Target Genes ◽  
Control Function ◽  
Beclin 1 ◽  
Dependent Manner ◽  
Neuronal Growth

Background: MicroRNAs (miRNAs) have an increasing functional role in some neurodegenerative diseases. Autophagy, the degradation of bulk protein in the cytoplasm, is the quality control function of protein and has a protective role in the survival of neural cells. miR-433 may play a regulatory role in neurodegenerative diseases. Many aspects underlying the mechanism of miR-433 in neural development and neurodegeneration are not clear.Methods: In this study, we established stable cell lines expressing miR-433 by infecting mouse hippocampal neural cell line (HT-22) cells with rLV-miR-433 and the control rLV-miR. Pre-miR-433 expression was analyzed using polymerase chain reaction (PCR). Mature miR-433 expression was measured using quantitative PCR (qPCR). The effect of miR-433 overexpression on cell proliferation was determined using a CCK-8 assay and flow cytometry. RNA interference was used to analyze the function of Cdk12 in mediating the effect of miR-433 on cell proliferation. The effect of miR-433 overexpression on cell apoptosis was determined by flow cytometry. Autophagy-related genes Atg4a, LC3B, and Beclin-1 were determined using qPCR, Western blot, or immunofluorescence. In addition, RNA interference was used to analyze the effect of Atg4a on the induction of autophagy. TargetScan 7.2 was used to predict the target genes of miR-433, and Smad9 was determined using qPCR.Results: Our results indicated that miR-433 increased the expression of Atg4a and induced autophagy by increasing the expression of LC3B-Ⅱ and Beclin-1 in an Atg4a-dependent manner. In addition, miR-433 upregulated the expression of Cdk12 and inhibited cell proliferation in a Cdk12-dependent manner and promoted apoptosis in HT-22 cells under the treatment of 10-hydroxycamptothecin.Conclusion: The results of our study suggest that miR-433 may regulate neuronal growth by promoting autophagy and attenuating cell proliferation. This might be a potential therapeutic intervention in neurodegenerative diseases.

scholarly journals ONC-212 (I-39), a Novel Inhibitor of the UPR, Is Cytotoxic and Cytostatic Against CLL Cells Under in Vitro Conditions That Mimic the Tumor Microenvironment

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 3145-3145
Author(s):  
Narjis Rizwan ◽  
Yandong Shen ◽  
Edwin Iwanowicz ◽  
Stephen P. Mulligan ◽  
Kyle R Crassini ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Flow Cytometry ◽  
Effective Treatment ◽  
Cell Line ◽  
Propidium Iodide ◽  
Cycle Arrest ◽  
Migratory Capacity ◽  
Ic50 Values ◽  
Cells Cultured

Abstract Introduction Despite the revolution in the treatment of chronic lymphocytic leukemia (CLL) over the past decade with the introduction of novel inhibitors targeting the B-cell receptor (BCR) signaling pathway and the Bcl-2 family of proteins, relapse is still common. Recent studies suggest that imipridones, a novel class of small molecule agents that attenuate mitochondrial respiration and modulate an immune response against cancer cells, may be an effective treatment option for several difficult to treat cancers. We investigated the effects of the imipridone, ONC-212 (I-39, first published by Nanjing Gator Meditech), as a potential therapeutic strategy for CLL using the OSU-CLL cell line and a modified OSU-CLL line in which TP53 was stably knocked out and primary CLL cells cultured under conditions that mimic the tumour microenvironment (TME). Methodology Primary CLL cells were co-cultured with CD40L-expressing fibroblasts to mimic aspects of the TME. The cytotoxicity of ONC-212 was assessed using the mitochondrial dye DiIC1(5), propidium iodide and flow cytometry. The effects of the drug on the adhesive and migratory capacity of primary CLL cells were evaluated using antibodies against CD49d, CXCR4 and an in vitro migration assay using stroma-derived factor 1a (SDF1-α). Changes in protein expression were assessed by immuno-blotting. The effects of ONC-212 on the cell cycle and proliferation were assessed using the OSU-CLL cell line. OSU-CLL cells were modified using the CRISPr-Cas9 technology to be TP53 deficient (OSU-TP53ko). The proportion of cells in each cycle phase was determined using propidium iodide and flow cytometry. Cell proliferation rates were determined using carboxyfluorescein succinimidyl ester (CFSE) and flow cytometry. Results ONC-212 induced apoptosis in a dose-dependent manner in primary CLL cells cultured in medium alone or in contact with CD40L-fibroblasts (Figure 1); the IC50 values were 72.97 nm +/- 1.45 nM and 472 +/- 2.04 nM, respectively. OSU-CLL and OSU-TP53ko cells were also sensitive to ONC-212, although the TP53 deficient line was less sensitive than OSU-CLL(Figure 1). IC50 values for the cell lines were 22 +/- 1.37 nM (OSU-CLL) and 48 +/- 3.25 nM (OSU-TP53ko). ONC-212 induced cell cycle arrest of the OSU-CLL and OSU-TP53ko lines at the G1/S phase transition. This effect was concomitant with a significant reduction in the proliferation of both lines. ONC-212 significantly down-regulated expression of the adhesion molecule CD49d and the G-coupled protein receptor CXCR4 on primary CLL cells. Down-regulation of CXCR4 translated into a decrease in the migratory capacity of CLL cells along an SDF1-α gradient. Immunoblotting suggested the mechanisms of action of ONC-212 include inhibition of ERK1/2-MAPK, a decrease in the Bcl-2/Bax ratio and upregulation of the pro-apoptotic Puma and Bak proteins. Conclusions ONC-212 is highly effective against CLL cells at nanomolar concentrations, against cells cultured under conditions that mimic aspects of the TME and against TP53-deficient cells. ONC-212 has cytotoxic effects, induces cell cycle arrest, slows proliferation and inhibits the mechanisms by which CLL cells migrate to and are retained within the TME. ONC-212 inhibited signaling downstream of the BCR and induced a pro-apoptotic 'tipping' of the balance in expression of BCl-2 family proteins. These data suggest ONC-212 may represent an effective treatment for CLL, particularly for patients who have high risk, relapsed/refractory disease associated with loss or mutation of TP53. Disclosures No relevant conflicts of interest to declare.

MLL-AF4 Down-Regulates CDKN1B (P27) Independent of Cell Cycle Progression.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 2563-2563
Author(s):  
Zhenbiao Xia ◽  
Relja Popovic ◽  
Tara Lorenz ◽  
Donna Santillan ◽  
Frank Erfurth ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Growth ◽  
Cell Line ◽  
Cell Cycle Progression ◽  
Target Genes ◽  
Leukemia Cell ◽  
Leukemia Cell Line ◽  
Luciferase Reporter ◽  
Cycle Progression ◽  
Downstream Target

Abstract The MLL gene, involved in many chromosomal translocations associated with acute myeloid and lymphoid leukemia, has more than forty known partner genes with which it is able to form in- frame fusions. MLL fusion genes transform hematopoietic cells in vitro, and cause leukemia in mouse models. However, the mechanism is still not clear. Characterizing important downstream target genes may provide rational therapeutic strategies for the treatment of MLL-associated leukemia. We explored potential downstream target genes of the most prevalent MLL fusion protein, MLL-AF4, which is primarily associated with pro-B ALL and is involved in the majority of infant leukemia. To this end, we developed an inducible MLL-AF4 fusion cell line. Overexpression of MLL-AF4 does not lead to increased proliferation in this cell line, but rather, cell growth is slowed compared to similar cell lines inducibly expressing truncated MLL. To try to understand the reason for slower cell growth, we assayed for expression of several CDK inhibitors. We found that in the MLL-AF4 induced cell line, the amount of CDKN1B (cyclin-dependent kinase inhibitor P27) was dramatically decreased both at the RNA and protein levels, in contrast, the levels of CDKN1A (P21) and CDKN2A (P16) were unchanged. Interestingly, we did not observe an increased percentage of cells in S phase of the cell cycle. To explore whether CDKN1B might be a direct target of MLL-AF4, we employed chromatin immunoprecipitation (ChIP) assays and luciferase reporter gene assays. We observed that MLL-AF4 binds to the CDKN1B promoter in vivo and represses CDKN1B promoter activity. Further, we confirmed CDKN1B promoter binding by ChIP assays in the MLL-AF4 leukemia cell line MV4-11. Our results suggest that the CDKN1B may be a downstream target of MLL-AF4, and that MLL-AF4 inhibits CDKN1B expression independent of cell cycle progression.

Bortezomib Can Overcome Stroma-Mediated Chemotherapy Resistance in Acute Lymphoblastic Leukemia.

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 2475-2475
Author(s):  
Ibraheem H. Motabi ◽  
Julie Ritchey ◽  
Matthew Holt ◽  
John F. DiPersio
Keyword(s):  
Cell Cycle ◽  
Flow Cytometry ◽  
Cell Line ◽  
Stromal Cells ◽  
Stromal Cell ◽  
S Phase ◽  
Cxcr4 Expression ◽  
Leukemic Cells ◽  
Apoptosis Rate ◽  
G2 Cells

Abstract Abstract 2475 Background: In spite of excellent results of chemotherapy induction resulting in high rates of complete remission in patients with ALL, relapse remains a major problem. This is likely due to a subset of leukemic cells that are chemotherapy resistant. The interaction between bone marrow stromal cells and leukemia cells protects leukemic cells from the cytotoxic effects of chemotherapy. Bortezomib is a proteozome inhibitor used to treat lymphoid malignancies. It has been shown to mobilize stem cells in myeloma patients. We hypothesized that, in addition to its direct cytotoxic effect, bortezomib can also overcome the protective effect of stromal cells by disrupting CXCR4 signaling through inhibition of CXCR4 turnover normally regulated by proteosome degradation. Methods: We used the human B-cell ALL cell lines, G2 and BV-173, and two human BM stromal cell lines, HS-5 and HS-27a for our studies. G2 cells were incubated alone or co-cultured with HS-5 or HS-27a for 24 hours then treated with RPMI (control), cytarabine, doxorubicin, or bortezomib. After 48 hours, cells were harvested and stained with FITC-conjugated anti-human annexin V antibody and analyzed by flow cytometry. To test the effect of bortezomib on CXCR4 expression, G2 cells were incubated with or without stromal cells and treated with cytarabine or bortezomib for 18 hours or left untreated. Then cells were stained with PE-conjugated anti-human CXCR4 antibody (clone 1D9) and analyzed by flow cyometry. The CXCR4 expression was reported as relative mean fluorescent intensity (RMFI) compared to isotype control. We then performed cell cycle analysis of G2 cells treated with cytarabine or bortezomib for 18 hours. All cells were treated with BrdU 1 hour prior to harvest, then stained with APC-conjugated anti-BrdU antibody and analyzed by flow cytometry. Results: Incubation of G2 cells with HS-5 protected them from apoptosis induced by cytarabine or doxorubicin but not bortezomib. The apoptosis rate for cytarabine treated cells was 55±0.3% without HS-5 vs. 15%±0.8 with HS-5(p<0.0001). The apoptosis rate of doxorubicin treated cells was 23±0.6% without HS-5 vs. 4.2±0.8% with HS-5 (p<0.0001). In the case of bortezomib, the apoptosis rate was 67±0.6% without HS-5 vs. 71±1.2% with HS-5. Similar results were observed with the BV-173 ALL cell line and with the use of HS-27a stromal cell line. We noted down-regulation of CXCR4 expression when G2 cells were co-cultured with stromal cells or after bortezomib treatment. In the absence of stromal cells, the CXCR4 RMFI was 40±3 in untreated cells vs. 16±0.4 in bortezomib treated cells (P=0.0015). In the presence of stromal cells CXCR4 RMFI 15±0.7% in untreated cells vs. 5±0.2% in bortezomib treated cells (P=0.0002). In cell cycle analysis, we found only 1.03±0.2% of cells treated with cytarabine were in S phase compared to 10.05±0.7% when cells were treated with bortezomib (P=0.0003). Conclusion: Bortezomib can overcome the stromal cell-mediated protection of human G2 ALL cells. This effect may be, in part, mediated by in the decreased surface expression of CXCR4 shown by others to mediate leukemia-stroma interaction and chemoprotection. In contrast to cytarabine which selectively kills cells in S phase, bortezomib induced killing of ALL cells regardless of cell cycle status. Future studies will use of in vivo mouse models of ALL to test the effects of bortezomib alone or in combination with chemotherapy on survival. Disclosures: DiPersio: genzyme: Honoraria.

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