Induction of Bim Facilitates Apoptosis in Leukemia Cells Treated with HDAC Inhibitors.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1994-1994
Author(s):  
Matthew C. Stubbs ◽  
Teresa Kim ◽  
Andrei Krivtsov ◽  
Peter Atadja ◽  
Scott A. Armstrong ◽  
...  
Keyword(s):  
Gene Expression ◽  
Histone Deacetylase ◽  
Cell Lines ◽  
Expression Patterns ◽  
Hdac Inhibitors ◽  
Leukemia Cell ◽  
Glucocorticoid Resistance ◽  
Leukemia Cell Line ◽  
Dependent Manner ◽  
Myeloid Leukemias

Abstract Lymphoblastic leukemias containing chromosomal translocations involving the Mixed Lineage Leukemia (MLL, HRX, ALL-1) gene, as well as most acute myeloid leukemias (AMLs) have relatively poor clinical prognoses due in part to intrinsic drug resistance. Therefore, new avenues are being explored for treatment of MLL-rearranged ALL and AMLs. One possible new therapeutic class currently being investigated is the histone deacetylase (HDAC) inhibitors. We utilized the histone deacetylase inhibitor NVP-LAQ824 (Novartis, Basel, Switzerland) and analyzed its effects on MLL rearranged and other myeloid leukemias. We also made use of an MLL-AF9 expressing myeloid leukemia cell line (AKLG) derived from purified murine leukemia stem cells to perform gene expression analysis on NVP-LAQ824 treated cells in order to further understand the mechanism of action of HDAC inhibitors, and to potentially identify cooperating therapeutics. NVP-LAQ824 inhibits cell growth at similar concentrations for all cell lines and primary patient samples tested (~25–50nM) as determined by MTT assay 48 hours after treatment. NVP-LAQ824 does not appear to induce apoptosis solely through inhibition of the HSP90/FLT3-ITD complex as cell lines possessing FLT3-ITD (a HSP90-chaperoned protein) and cells without this mutation have similar drug sensitivities. In fact, in cells overexpressing FLT3-ITD that are treated with NVP-LAQ824, phospho-FLT3-ITD levels do not diminish. Microarray data indicated that NVP-LAQ824 induces the BH3-only family member bim. This finding was verified by Western blotting in all cell lines and patient samples tested. Further, shRNA-mediated knockdown of Bim induced relative resistance of cells to NVP-LAQ824. The expression profile also showed similarities to gene expression patterns of dexamethasone treated cells, namely, increased bim levels and decreased expression of c-myc, raising the possibility of synergy between the two drugs. Using MTT assays, we discovered that NVP-LAQ824 in low doses (25nM) induces sensitivity to dexamethasone in glucocorticoid resistant cell lines in a glucocorticoid receptor (GR) dependent manner. Therefore, our data indicate that NVP-LAQ824 may reverse glucocorticoid resistance and may provide insight into glucocorticoid resistance in MLL rearranged leukemias. The biochemistry behind HDAC inhibitors merits further study.

Analysis of Class I and II Histone Deacetylase Fails To Identify a Human Leukemia Specific Expression Profile.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 2130-2130 ◽  
Author(s):  
Guillermo Garcia-Manero ◽  
Andy S. Quesada ◽  
Shirisha Maddipoti ◽  
Shaoqing Kuang ◽  
Weigang Tong ◽  
...  
Keyword(s):  
Clinical Trials ◽  
B Cells ◽  
B Cell ◽  
Histone Deacetylase ◽  
Cell Lines ◽  
Expression Profiles ◽  
Hdac Inhibitors ◽  
Leukemia Cell ◽  
Leukemia Cell Lines ◽  
Normal Controls

Abstract Histone deacetylase (HDAC) inhibitors are being developed clinically for the treatment of leukemia. Because HDACs are composed of a large number of different proteins, and substrate specificity may differ among different HDAC inhibitors, it is important to understand if human leukemias are characterized by specific HDAC expression patterns. To study this, we have analyzed using real-time PCR and Western blots, all major class I and II HDACs in normal marrow controls (N=13, including 10 CD19+ B cell specimens), leukemia cell lines (N=25), samples from patients with AML (N=6), MDS (N=12), CLL (N=10) and human samples (N=6) obtained pretreatment and sequentially from patients with leukemia treated with two different phase I clinical trials of HDAC inhibitors: MGCD0103 and vorinostat. In general, normal controls were characterized by low levels of HDACs 1 to 10, although normal CD19+ B cells exhibited a significant increased expression of HDAC1 and 5. In leukemia cell lines, HDAC 1, 2 and 3 were expressed at higher levels than 4 to 10 but there were no differences between leukemia cell lines and normal controls or B cells. HDAC mRNA expression was not modified by cell proliferation or treatment with HDAC inhibitors. No specific HDAC expression profiles were detected in primary human AML or MDS samples. In contrast, CLL primary samples were characterized by an overexpression of HDAC 1,3,5 and 10, although this pattern was not significantly different than that of normal CD19+ B cells. Sequential analysis of human samples obtained from patients treated with two different HDAC inhibitors, vorinostat or MGCD0103 on two different clinical trials, did not affect expression profiles in patients with MDS or AML. Overall, mRNA expression results correlated with protein levels. In summary, our results indicate that AML or MDS are not characterized by a leukemia specific HDAC expression profile but that B cells and B cell leukemia are characterized by a significant overexpression of HDAC 1. This could explain the activity observed with HDAC inhibitors in B cell malignancies and serve as the bases for clinical studies of HDAC inhibitors in CLL.

scholarly journals Effect of tamoxifen on cell lines displaying the multidrug-resistant phenotype [see comments]

Blood ◽  
1991 ◽  
Vol 77 (4) ◽  
pp. 818-825 ◽  
Author(s):  
E Berman ◽  
M Adams ◽  
R Duigou-Osterndorf ◽  
L Godfrey ◽  
B Clarkson ◽  
...  
Keyword(s):  
Cell Line ◽  
Cell Lines ◽  
Leukemia Cell ◽  
Multidrug Resistant ◽  
Leukemia Cell Line ◽  
Parent Cell ◽  
Continuous Exposure ◽  
Dependent Manner ◽  
Uptake Pattern ◽  
Dose Dependent

Abstract We examined the effect of tamoxifen (Tmx), verapamil, and daunorubicin (DNR) in two cell lines that displayed the multidrug-resistant (MDR) phenotype and used laser flow cytometry to quantitate intracellular DNR content. In the vinblastine-resistant human lymphoblastic lymphoma cell line CEM-VBL, simultaneous incubation of DNR with Tmx 10 mumol/L or Tmx 50 mumol/L increased intracellular DNR fluorescence in a dose-dependent manner and demonstrated an uptake pattern similar to that seen with DNR and verapamil. Similar results were obtained in the vincristine- resistant human myeloid leukemia cell line HL-60/RV+. Cellular retention of DNR was also measured in both cell lines and results suggested that continuous exposure of the cells to Tmx resulted in higher intracellular DNR content compared with cells resuspended in fresh medium. No effect of Tmx or verapamil was observed in the drug- sensitive parent cell lines CEM or HL-60. Clonogenic experiments were then performed to determine whether Tmx was itself inhibitory to cell growth or whether Tmx potentiated DNR cytotoxicity. Tmx 10 mumol/L did not significantly inhibit either CEM-VBL or HL-60/RV+ cells after a 3- hour exposure followed by culture in methylcellulose. Tmx 50 mumol/L was significantly more inhibitory in both cell lines. However, cells that had been incubated with DNR and Tmx 10 mumol/L demonstrated a marked increment in growth inhibition compared with cells that had been incubated with DNR alone or Tmx 10 mumol/L alone. Based on the data presented here, we suggest that clinical testing of Tmx and DNR be pursued in the setting where MDR may play a role.

To Study the Proliferative Inhibition of Anticancer Drug in Two Kinds of Ph (+) Leukemia Cell Lines.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 4110-4110
Author(s):  
Yuping Gong ◽  
Xi Yang ◽  
Ting Niu
Keyword(s):  
Gene Expression ◽  
Acute Lymphoblastic Leukemia ◽  
Cell Line ◽  
Cell Lines ◽  
K562 Cell ◽  
Lymphoblastic Leukemia ◽  
Leukemia Cell ◽  
Myelogenous Leukemia ◽  
Leukemia Cell Line ◽  
Leukemia Cell Lines

Abstract Abstract 4110 Objective To study the proliferative inhibition of imatinib, daunorubicin and bortezomib in two kinds of Ph(+) leukemia cell lines: chronic myelogenous leukemia cell line K562 expressing P210 protein and acute lymphoblastic leukemia cell line SUP-B15 expressing P190 protein. Methods (1) Cell proliferation with imatinib, daunorubicin and bortezomib for 72 hours was analyzed by the MTT assay and displayed by growth curve and IC50 value. (2) The change of bcr-abl gene mRNA levels after the 48 hours' intervention of imatinib (final concentration at 0μM, 0.35μM, 1 μM) was detected by reverse transcription polymerase chain reaction (RT-PCR). Results (1) The IC50 values of K562 and SUP-B15 cells inhibited by imatinib, daunorubicin and bortezomib for 72 hours was respectively 0.286±0.06 (μmol/L), 0.303±0.009 (μmol/L), 22.127±3.592 (nmol/L) and 1.387±0.180(μmol/L), 0.117±0.017 (μmol/L), 12.350±0.740 (nmol/L), which indicated that the K562 cell line was the more sensitive to imatinib than SUP-B15 cell line, whereas the SUP-B15 cell line had the more sensitivity to daunorubicin and bortezomib. (2) There was no change of bcr-abl gene expression after the 48 hours' intervention of imatinib in both cell lines. Conclusion (1) Imatinib, daunorubicin and bortezomib had good anti-cancer effect to Ph+ leukemia cells in vitro. What's more, the K562 cell was the more sensitive to imatinib and only imatinib will have good effect on chronic myelogenous leukemia. Whereas the SUP-B15 cell had the more sensitivity to daunorubicin and bortezomib and combining imatinib with daunorubicin or bortezomib, the effect will be better on Ph(+) acute lymphoblastic leukemia. (2) The short time intervention of imatinib had no effect on the bcr-abl gene expression and imatinib could need long time to show curative effect for the Ph+ leukemia. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Deep sequencing reveals two Jurkat subpopulations with distinct miRNA profiles during camptothecin-induced apoptosis

2017 ◽  
Author(s):  
İpek Erdoğan ◽  
Mehmet İlyas Coşacak ◽  
Ayten Nalbant Aldanmaz ◽  
Bünyamin Akgül
Keyword(s):  
Gene Expression ◽  
T Cells ◽  
Cell Lines ◽  
Deep Sequencing ◽  
Mirna Expression ◽  
Expression Profiles ◽  
Leukemia Cell ◽  
Jurkat Cells ◽  
Cellular Heterogeneity ◽  
Leukemia Cell Line

AbstractmicroRNAs (miRNAs) are small non-coding RNAs of about 19-25 nt that regulate gene expression post-transcriptionally under various cellular conditions, including apoptosis. The miRNAs involved in modulation of apoptotic events in T cells are partially known. However, heterogeneity associated with cell lines makes it difficult to interpret gene expression signatures especially in cancer-related cell lines. Treatment of Jurkat T cell leukemia cell line with the universal apoptotic drug, camptothecin, resulted in identification of two Jurkat sub-populations: one that is sensitive to camptothecin and the other being rather intrinsically resistant. We sorted apoptotic Jurkat cells from the non-apoptotic ones prior to profiling miRNAs through deep sequencing. Our data showed that a total of 184 miRNAs were dysregulated. Interestingly, apoptotic and non-apoptotic sub-populations exhibited a distinct miRNA expression profile. In particular, 6 miRNAs were inversely expressed in these two sub-populations. The pyrosequencing results were validated by real time qPCR. Altogether these results suggest that miRNAs modulate apoptotic events in T cells and that cellular heterogeneity requires careful interpretation of miRNA expression profiles obtained from drug-treated cell lines.

scholarly journals Histone deacetylase (HDAC) encoding gene expression in pancreatic cancer cell lines and cell sensitivity to HDAC inhibitors

2008 ◽  
Vol 7 (4) ◽  
pp. 523-531 ◽  
Author(s):  
M. Ouaïssi ◽  
S. Cabral ◽  
J. Tavares ◽  
A. Cordeiro da Silva ◽  
F. Mathieu-Daude ◽  
...  
Keyword(s):  
Gene Expression ◽  
Pancreatic Cancer ◽  
Histone Deacetylase ◽  
Cell Lines ◽  
Cancer Cell ◽  
Pancreatic Cancer Cell ◽  
Hdac Inhibitors ◽  
Cancer Cell Lines ◽  
Cell Sensitivity ◽  
Encoding Gene

Novel Synthetic Histone Deacetylase Inhibitor, SK-7041, Has Potent Anti-Proliferative Activity in Acute Myeloid Leukemia Cell Lines.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 2854-2854
Author(s):  
Sung-Soo Yoon ◽  
Eunkyung Bae ◽  
Juwon Park ◽  
Yongjun Cha ◽  
Young Y. Lee ◽  
...  
Keyword(s):  
Gene Expression ◽  
Histone Deacetylase ◽  
Cell Lines ◽  
Histone Deacetylase Inhibitors ◽  
Expression Profiles ◽  
Leukemia Cell ◽  
Gene Expression Profiles ◽  
Myelogenous Leukemia ◽  
G1 Arrest ◽  
Cancer Cell Growth

Abstract Histone acetyltransferase (HAT) and histone deacetylase (HDAC) activities determine the acetylation status of histones, which regulates gene expression through chromatin remodeling. Aberrant histone acetylation is known to play a key role in leukemogenesis. Thus, histone deacetylase inhibitors (HDACIs) are emerging as a new class of anti-cancer agents for leukemia. In this study, we examined anti-proliferative effects of novel hybrid synthetic HDACI, SK-7041, in various acute myelogenous leukemia (AML) cell lines (KG-1, HL-60, HEL, U937, ML-1). SK-7041 induced the time-dependent hyperacetylation of histones H3 and H4 in AML cell lines. It preferentially inhibited the enzymatic activities of HDAC1 and HDAC2, as compared with the other HDAC isotypes, indicating that class I HDAC is the major target of SK-7041. All the cell lines tested showed similar patterns of growth inhibition by SK-7041. Their IC50 values were approximately 0.5 mM at 72 hours incubation. SK-7041 effectively induced the apoptosis via activation of caspase-3, -7, -9, and PARP, but not caspase-8. SK-7041 enhanced G1 arrest via decreasing Cyclin D1 expression and increasing p21 expression. Changes in gene expression profiles after treating KG-1 cells with various concentrations of SK-7041 were assessed using a cDNA microarray consisting of distinct cDNAs of cancer-related genes. 7 genes, namely RGL1, FYN, CARD9, ABCA7, TNFRSF6B, CASP9, and ENPP2 were induced substantially (Global M >2.0) and 8 genes, namely PTPN7, CD34, INSIG1, IL16, LHX6, TRIB3, BID, and PDCD4 were reduced substantially (Global M < 2.0). In conclusion, this study showed SK-7041 inhibited cancer cell growth and caused characteristic gene expression profile changes in AML cells. The alteration in levels of acetylated histones was closely associated with expression of specific cancer-related genes in AML cells.

Chromatin immunoprecipitation (ChIP) studies indicate a role for CCAAT enhancer binding proteins alpha and epsilon (C/EBPα and C/EBPε) and CDP/cut in myeloid maturation-induced lactoferrin gene expression

Blood ◽  
2003 ◽  
Vol 101 (9) ◽  
pp. 3460-3468 ◽  
Author(s):  
Arati Khanna-Gupta ◽  
Theresa Zibello ◽  
Hong Sun ◽  
Peter Gaines ◽  
Nancy Berliner
Keyword(s):  
Gene Expression ◽  
Chromatin Immunoprecipitation ◽  
Leukemia Cell ◽  
Myeloid Cells ◽  
In Vitro Models ◽  
Leukemia Cell Line ◽  
Dependent Manner ◽  
Late Gene Expression ◽  
Ccaat Displacement Protein

In vitro models of granulopoiesis involving the inducible expression of either CCAAT enhancer binding protein alpha (C/EBPα) or C/EBPε in myeloid cells have been shown to lead to the induction of a granulocytic maturation program accompanied by the expression of myeloid-specific genes. Since members of the C/EBP family of transcription factors recognize and bind to similar DNA-binding motifs, it has been difficult to elucidate the specific role of each of the C/EBP family members in eliciting myeloid gene expression. In order to address this issue, we focused on the expression of the lactoferrin (LF) gene. LF expression is transcriptionally regulated in a C/EBP-dependent manner in myeloid cells. Using chromatin immunoprecipitation (ChIP) analysis we demonstrate that C/EBPα binds to the LF promoter in nonexpressing cells. Upon induction of maturation, C/EBPε binds to the LF promoter, which correlates with LF expression. Lack of LF expression in the acute promyelocytic leukemia cell line NB4, which harbors the t(15;17) translocation, cannot be correlated with aberrant binding at the C/EBP site in the LF promoter. It is, however, associated with the persistent binding of the silencer CCAAT displacement protein (CDP/cut) to the LF promoter in these cells. We conclude that C/EBPα, C/EBPε, and CDP/cut all play definitive roles in regulating late gene expression during normal myeloid development.

scholarly journals Human eosinophil Charcot-Leyden crystal protein: cloning and characterization of a lysophospholipase gene promoter

Blood ◽  
1993 ◽  
Vol 82 (6) ◽  
pp. 1868-1874 ◽  
Author(s):  
HI Gomolin ◽  
Y Yamaguchi ◽  
AV Paulpillai ◽  
LA Dvorak ◽  
SJ Ackerman ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Lines ◽  
Binding Sites ◽  
Transcriptional Start Site ◽  
Leukemia Cell ◽  
Leukemia Cell Line ◽  
Luciferase Expression ◽  
Mrna Levels ◽  
Start Site ◽  
Human Eosinophil

Abstract The Charcot-Leyden crystal (CLC) protein is a lysophospholipase expressed exclusively by eosinophils and basophils. During eosinophilic differentiation of eosinophil-committed cell lines, CLC steady state mRNA levels increase significantly. This increased expression is transcriptionally regulated during butyrate induction of an eosinophilic subline (C15) of the promyelocytic leukemia cell line HL- 60, as shown by nuclear run-on assays. The transcriptional start site of the CLC gene was identified 43 bp upstream of the 5′ end of the longest available cDNA sequence. The gene encoding CLC protein was cloned from a chromosome 19-specific library and a fragment overlapping the transcriptional start site was isolated and sequenced. Plasmid constructs (in the pXP2 luciferase expression vector) containing 411 and 292 bp of genomic sequence upstream of the CLC transcriptional start site directed reporter gene expression in transient transfections of HL-60-C15 cells, as well as other myeloid (U937) and nonmyeloid (HeLa and RPMI 8402) cell lines. However, the differential expression of the two CLC promoter constructs in these cell lines suggests that the -292 to -411 bp region of the promoter may confer some specificity for expression in the eosinophil lineage. The CLC promoter sequence contains two consensus GATA binding sites, a purine-rich sequence that presents potential binding sites for PU.1, a member of the ets family of genes, as well as sequences described in other myeloid-specific promoters. This is the first demonstration of a functional eosinophil promoter that could serve as a model for identifying DNA elements and trans-activating factors that regulate gene expression during the commitment and differentiation of the eosinophil lineage.

scholarly journals Effect of tamoxifen on cell lines displaying the multidrug-resistant phenotype [see comments]

Blood ◽  
1991 ◽  
Vol 77 (4) ◽  
pp. 818-825
Author(s):  
E Berman ◽  
M Adams ◽  
R Duigou-Osterndorf ◽  
L Godfrey ◽  
B Clarkson ◽  
...  
Keyword(s):  
Cell Line ◽  
Cell Lines ◽  
Leukemia Cell ◽  
Multidrug Resistant ◽  
Leukemia Cell Line ◽  
Parent Cell ◽  
Continuous Exposure ◽  
Dependent Manner ◽  
Uptake Pattern ◽  
Dose Dependent

We examined the effect of tamoxifen (Tmx), verapamil, and daunorubicin (DNR) in two cell lines that displayed the multidrug-resistant (MDR) phenotype and used laser flow cytometry to quantitate intracellular DNR content. In the vinblastine-resistant human lymphoblastic lymphoma cell line CEM-VBL, simultaneous incubation of DNR with Tmx 10 mumol/L or Tmx 50 mumol/L increased intracellular DNR fluorescence in a dose-dependent manner and demonstrated an uptake pattern similar to that seen with DNR and verapamil. Similar results were obtained in the vincristine- resistant human myeloid leukemia cell line HL-60/RV+. Cellular retention of DNR was also measured in both cell lines and results suggested that continuous exposure of the cells to Tmx resulted in higher intracellular DNR content compared with cells resuspended in fresh medium. No effect of Tmx or verapamil was observed in the drug- sensitive parent cell lines CEM or HL-60. Clonogenic experiments were then performed to determine whether Tmx was itself inhibitory to cell growth or whether Tmx potentiated DNR cytotoxicity. Tmx 10 mumol/L did not significantly inhibit either CEM-VBL or HL-60/RV+ cells after a 3- hour exposure followed by culture in methylcellulose. Tmx 50 mumol/L was significantly more inhibitory in both cell lines. However, cells that had been incubated with DNR and Tmx 10 mumol/L demonstrated a marked increment in growth inhibition compared with cells that had been incubated with DNR alone or Tmx 10 mumol/L alone. Based on the data presented here, we suggest that clinical testing of Tmx and DNR be pursued in the setting where MDR may play a role.

scholarly journals Human eosinophil Charcot-Leyden crystal protein: cloning and characterization of a lysophospholipase gene promoter

Blood ◽  
1993 ◽  
Vol 82 (6) ◽  
pp. 1868-1874 ◽  
Author(s):  
HI Gomolin ◽  
Y Yamaguchi ◽  
AV Paulpillai ◽  
LA Dvorak ◽  
SJ Ackerman ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Lines ◽  
Binding Sites ◽  
Transcriptional Start Site ◽  
Leukemia Cell ◽  
Leukemia Cell Line ◽  
Luciferase Expression ◽  
Mrna Levels ◽  
Start Site ◽  
Human Eosinophil

The Charcot-Leyden crystal (CLC) protein is a lysophospholipase expressed exclusively by eosinophils and basophils. During eosinophilic differentiation of eosinophil-committed cell lines, CLC steady state mRNA levels increase significantly. This increased expression is transcriptionally regulated during butyrate induction of an eosinophilic subline (C15) of the promyelocytic leukemia cell line HL- 60, as shown by nuclear run-on assays. The transcriptional start site of the CLC gene was identified 43 bp upstream of the 5′ end of the longest available cDNA sequence. The gene encoding CLC protein was cloned from a chromosome 19-specific library and a fragment overlapping the transcriptional start site was isolated and sequenced. Plasmid constructs (in the pXP2 luciferase expression vector) containing 411 and 292 bp of genomic sequence upstream of the CLC transcriptional start site directed reporter gene expression in transient transfections of HL-60-C15 cells, as well as other myeloid (U937) and nonmyeloid (HeLa and RPMI 8402) cell lines. However, the differential expression of the two CLC promoter constructs in these cell lines suggests that the -292 to -411 bp region of the promoter may confer some specificity for expression in the eosinophil lineage. The CLC promoter sequence contains two consensus GATA binding sites, a purine-rich sequence that presents potential binding sites for PU.1, a member of the ets family of genes, as well as sequences described in other myeloid-specific promoters. This is the first demonstration of a functional eosinophil promoter that could serve as a model for identifying DNA elements and trans-activating factors that regulate gene expression during the commitment and differentiation of the eosinophil lineage.

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