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.

Exploiting Endogenous Micro-RNAs to Avoid off-Target Transgene Expression

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 3296-3296
Author(s):  
Raul Teruel Montoya ◽  
Xianguo Kong ◽  
Shaji Abraham ◽  
Lin Ma ◽  
Leonard C. Edelstein ◽  
...  
Keyword(s):  
Gene Expression ◽  
T Cells ◽  
Cell Lines ◽  
Binding Sites ◽  
Transgene Expression ◽  
Cell Types ◽  
Jurkat Cells ◽  
Cell Type ◽  
Hematological Disorders

Abstract Abstract 3296 Genetic modification of hematopoietic stem cells (HSCs) has the potential to benefit acquired and congenital hematological disorders. Despite the use of so-called “tissue-specific” promoters to drive expression of the desired transgene, off-target (and consequent deleterious) effects have been observed. MicroRNAs (miRNAs) are important regulators of gene expression. They associate with Argonaute proteins and most typically target 3'UTRs, where complementary base-pairing results in repressed gene expression via RNA decay and translation inhibition. Most miRNAs are ubiquitously expressed, and although some are claimed to be “tissue specific,” such claims have generally not been rigorously validated. The long-term goal of this work is identifying “cell preferential” miRNA expression that could be exploited in expression vectors to minimize off-target transgene expression in HSCs. Initially, total RNA was extracted with Trizol from the megakaryocyte and T-lymphocyte cell lines, Meg-01 and Jurkat, and miRNAs were profiled by Nanostring technology (Nanostring Technologies, Denver, CO). MiR-495 was determined to be highly expressed in Meg-01 and very low in Jurkat cells. A luciferase reporter construct was generated with four canonical binding sites for miR-495 in the 3'UTR and transfected into both cell lines. Compared to control vector without miR-495 binding sites, luciferase expression showed a 50% reduction in Meg-01 cells, but no knock down in Jurkat cells. These experiments indicated that different levels of endogenous miRNA levels can regulate transgene expression through a novel design in the 3'UTR. We next turned our attention to human hematopoietic cells. We reasoned that the long-term goal of minimal off-target transgene expression in HSCs would require knowledge of miRNAs that had little or no detectable expression (“selectively reduced [SR]”) in one cell type and were highly expressed in other cell types. In this manner, the transgene expression would be dampened only in the non-target cells. As a surrogate for bone marrow progenitors and as proof of principle, we used primary cells in normal human peripheral blood. T-cells, B-cells, platelets and granulocytes were purified by density centrifugation followed by immunoselection from five healthy human donors. Flow cytometry using membrane specific markers demonstrate >97% purity of each specific cell preparation. Total RNA was extracted and miRNAs were profiled as above. First, we identified 277 miRNAs that were differentially expressed between any pair of cell types (p-value<0.05 by ANOVA). Second, we performed ranked pair-wise comparisons across all cell types to determine SR miRNAs. This analysis revealed 5 platelet SR-miRNAs, 6 B-cell SR-miRNAs, 2 T-cell SR-miRNAs and 4 granulocyte SR-miRNAs. Lastly, we considered which of these 17 SR-miRNAs would be the best single SR-miRNA within and across cell types. SR-miRNAs were normalized to let-7b, a miRNA we determined to be equivalently expressed across all cell types, and hence, an ideal normalizer. Lineage-specific SR-miRNAs were selected based on extremely low expression in only one cell type and highest fold change of expression compared to the other cell types. The best SR-miRNAs were miR-29b (SR in platelets), miR-125a-5p (SR in B-cells) and miR-146a (SR in granulocytes). The SR expression levels of these 3 miRNAs were validated by qRT-PCR. Our analysis identified no good SR-miRNAs in T-cells. On-going experiments are testing the selective effects of the SR miRNAs in lentiviral vector infection of cord blood CD34+ cells differentiated along specific lineages. In summary, we have demonstrated in hematopoietic cell lines that SR endogenous miRNAs can regulate the expression of transgenes via tandem arrangement of their target sites in the 3'UTR. Additionally, we have identified miRNAs that are specifically expressed at a very low level in one blood cell type and at high levels in other cell types. These miRNAs could potentially be utilized as new biological tools in gene therapy for hematological disorders to restrict transgene expression and avoid the negative consequences of off-target expression. Disclosures: No relevant conflicts of interest to declare.

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.

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.

Study on Leukemia Cell Differentiation and Drug Resistance By SATB1 Gene

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 5172-5172
Author(s):  
Chunyan Wang ◽  
Huo Tan ◽  
Liu Lei ◽  
Lihua Xu
Keyword(s):  
Gene Expression ◽  
Drug Resistance ◽  
Cell Differentiation ◽  
Western Blot ◽  
Cell Lines ◽  
Jurkat Cell ◽  
Leukemia Cell ◽  
Jurkat Cells ◽  
Leukemia Cells ◽  
Chemotherapy Drugs

Abstract Background and objective: In this article, four types of lymphoma cell lines U937, Raji, Hut-102, Akata, and three types of leukemia cell lines K562, HL-60, Jurkat were used the expression of SATB1 in leukemia cells, Through cell differentiation induced by all-TRANS Retinoic acid and DMSO, finds the SATB1 gene expression is reduced, suggest that the SATB1 gene may be involved in cell differentiation. By gene silencing, reduce the SATB1 gene expression, sensitivity to chemotherapy drugs is increased, suggest that the SATB1 gene may be associated with drug resistance. Methods: 1. Western Blot Assay for detection of SATB1 gene expression in cell lines. 2. DMSO and ATRA to HL-60, Jurkat cells for different times, SATB1 gene expression in the cells are detected by Western Blot. 3. Building SATB1-shRNA, transfected Jurkat cells and to HL-60. 4. Testing inhibition rate of chemotherapy drugs on HL-60, HL-60-SATB1-ctr, HL-60-SATB1-sh, Jurkat, Jurkat-SATB1-ctr, Jurkat-SATB1-sh cell, suggesting its relationship with the drug resistance. Results: 1. SATB1 gene was expression in all the plant cells. 2. The expression of SATB1 in U937, Raji, Hut-102, Akata four types of lymphoma cell line is low. 3. It was highly expression in HL-60, Jurkat cells, but low in K562. 4. DMSO and ATRA treat HL-60, Jurkat cells for 48 and 96 hours, HL-60 cells become larger, rounded, Jurkat cells into smaller, is more obvious for 96-hour. 5. For HL-60, Jurkat cells, after DMSO and ATRA treated, the expression of SATB1 was decreased, more obvious for 96-hour. 6. HL-60-SATB1-sh1, Jurkat-SATB1-sh1 cell compared with the control group, the SATB1 protein content decreased significantly, successfully building SATB1-shRNA HL-60 and Jurkat cell line. 7. HL-60-SATB1-sh1, and Jurkat-SATB1-sh1 of daunorubicin (DNR) sensitivity has improved significantly. Conclusion: 1. The SATB1 gene expression in leukemia and lymphoma cells, and higher expression in leukemia cells. 2. After DMSO and ATRA treatment, the SATB1 expression significantly reduced, suggest that SATB1 may be involved in cell differentiation. 3. Successfully built SATB1-shRNA, and successfully transferred to HL-60 and Jurkat cell lines. 4. HL-60-SATB1-sh1, and Jurkat-SATB1-sh1 of daunorubicin (DNR) sensitivity increased significantly, prompting SATB1 may be related to drug-resistance. Disclosures No relevant conflicts of interest to declare.

Anti-invasive and antiproliferative effects of Pleurotus ostreatus extract on acute leukemia cell lines

2018 ◽  
Vol 29 (1) ◽  
pp. 95-102 ◽  
Author(s):  
Alireza Ebrahimi ◽  
Amir Atashi ◽  
Masoud Soleimani ◽  
Maedeh Mashhadikhan ◽  
Ahmadreza Barahimi ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Lines ◽  
Pleurotus Ostreatus ◽  
Leukemia Cell ◽  
Jurkat Cells ◽  
Antiproliferative Effects ◽  
Invasion And Migration ◽  
Natural Medicine ◽  
Leukemia Cell Lines ◽  
And Migration

AbstractBackground:Currently, mushrooms have been used in traditional and folk medicines for their therapeutic activities, such as antibiotic, antitumor, anti-inflammatory, anticancer, antileukemic and immunomodulatory actions. This investigation evaluates the anti-invasive, antiproliferative and cytotoxic effects ofPleurotus ostreatus(Pleurotaceae) on leukemia cell lines.Methods:The proliferation of KG-1 cells was measured by using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay after treatment with gradient dilutions ofP. ostreatusextract. Then, the minimum inhibitory concentration (MIC) of the extract was determined. Moreover, the proliferation of Jurkat cells and bone marrow mesenchymal stem cells (BMSCs), a cancerous cell line and normal body cells, respectively, was considered. The apoptotic morphology of treated KG-1 cells was evaluated with Giemsa staining. The invasion and migration of cells were evaluated using transwell invasion assay. Thereafter, the rates of apoptosis and necrosis were measured by using flow cytometry, andBAXandMMP-9gene expression were evaluated using quantitative reverse transcription-polymerase chain reaction as apoptotic and metastatic genes, respectively.Results:The MIC of the extract was determined to be 1 mg/mL after 48 h. According to the results, the extract decreased the proliferation of leukemia cell lines (KG-1 and Jurkat cells) but had no antiproliferative effects on BMSCs. Moreover, KG-1 cell migration andMMP-9gene expression decreased after the treatment, and the rate of apoptosis andBAXgene expression increased significantly.Conclusions:According to the efficient therapeutic properties ofP. ostreatuson leukemia cell lines, this mushroom could be introduced as a natural medicine to cure leukemic patients who suffer from the harmful side effects and enormous costs of chemotherapy.

scholarly journals Generating AML-Specific Peripheral Blood Autologous Cytotoxic T-Lymphocytes (CTLs)

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 5806-5806
Author(s):  
Rohtesh S. Mehta ◽  
Xiaohua Chen ◽  
Antony Jeyaraj ◽  
Paul Szabolcs
Keyword(s):  
Gene Expression ◽  
T Cells ◽  
T Cell ◽  
Gene Expression Profile ◽  
Cell Lines ◽  
Expression Profile ◽  
Leukemia Cell ◽  
P Value ◽  
Cell Gene Expression ◽  
Cell Gene

Abstract Background: Ex-vivo expansion of CBT-cells using CD3/CD28 co-stimulatory beads, IL-2 + IL-7 and subsequent priming against leukemia cell lines using IL-15 generated specific CTLs. [1, 2] Hypothesis: We hypothesized that (a) patient-derived AML-specific PB auto CTLs could be generated with immune-stimulatory culture condition (b) Resistant AML samples would possess gene expression profiles similar to MDSCs (myeloid-derived suppressor cells) (c) Frequency of Tregs (CD4+CD25brightFoxP3+) and T-cell co-signaling molecules gene expression will be different between effective and ineffective CTLs. Methods: AML & auto T-cells were purified from cryopreserved PBMC of AML patients admitted with acute blast crisis (n=8). AML blasts were sustained in StemSpan™ Serum-Free media [STEMCELL Technologies] with MSC support + cytokine cocktail (IL-3, SCF, FLT3L, GMCSF, IL-4). T-cells were expanded in culture for 2 weeks as reported [1, 2] and subsequently primed with γ-irradiated auto AML weekly X 3 with IL15 + CD28ab [BD Biosciences]. At the end of week 3 (EOW3), cytotoxicity was assessed against AML and irrelevant targets - IM9 (lymphoid) and U937 (myeloid) cell lines, loaded with BATDA at an E:T ratio of 40:1, 20:1, 10:1 and 5:1 using DELFIA® EuTDA assay.[2] IFN-γ ELISPOT assay against same targets was also done.[2] RT-qPCR analysis was performed on AML & T-cells before and after priming, using Power SYBR® Green master mix (Thermo Fisher Scientific) and StepOne Plus system [Life Technologies]. Two-tailed student t-testcompared experimental groups. Results · T-cells expanded in all samples (n=8) with a median expansion of 155-fold (range 11-489), at EOW3. · ELISPOT assay was positive in 4/8 samples. [Fig 1] · CTL assay was difficult to standardize for primary AML blasts due to high degree of spontaneous apoptosis (>30% spontaneous release [SR]). · 2/8 samples were deemed evaluable (SR<30%). · Both samples showed AML-specific lysis. [Fig 2] · Overall, AML-specific autologous CTL could be generated from 5 of 8 samples based on ELISPOT & CTL assays, regardless of original FAB immunophenotype, not shown. · Tregs proportion declined significantly in effective CTLs post-priming as compared to pre-priming (56% to 24%, p-value 0.046, n=4). [Fig 3] · T-cell gene expression profiling showed significant differences in effective vs ineffective CTLs. [Table 1] · Resistant AML (n=3) had up-regulated downstream markers associated with MDSC generation compared to “non-resistant” AML (n=5). [Table 2] Conclusions (a) AML-specific auto CTLs can be generated (b) Tregs decreased with priming in effective CTLs (c) differential T-cell gene expression profile exists between effective and ineffective CTLs (d) AML gene expression suggests MDSC-like profile in resistant samples.Abstract 5806. TABLE 1:T-CELL GENE EXPRESSION PROFILE (POST VS PRE-PRIMING)Effective CTLs (n=5)Ineffective CTLs (n=3)GeneΔΔ Ct(Post - Pre) (mean, SEM)P-valueFold change (mean, SEM)ΔΔ Ct(Post - Pre) (mean, SEM)P-valueFold change (mean, SEM)4-1BB-3.17 (0.76)0.02514 (7.7)1.98 (1.04)0.190.39 (0.22)HVEM-2.43 (0.61)0.0287.3 (3.7)0.14 (1.65)0.951.57 (1.28)LIGHT-3.62 (0.73)0.01617.3 (7.3)1.78 (1.84)0.441.1 (0.98)PRKC-α-2.03 (0.47)0.0234.6 (1.1)1.89 (0.36)0.0340.29 (0.08)PRKC-θ-3.36 (0.59)0.0113.7 (6.7)0.25 (0.59)0.710.99 (0.41)LAIR1-3.81 (0.42)0.00316.2 (5.6)-1.35 (2.20)0.6017.15 (16.5)PP2A-2.40 (0.57)0.0256.7 (2.6)0.49 (1.57)0.791.89 (1.52)2B4-1.53 (1.14)0.274.98 (1.82)-3.48 (0.11)0.0211.2 (0.9)LTA-α-1.18 (0.78)0.233.61 (2.11)2.69 (0.18)0.0430.16 (0.02)LTA-β-0.93 (0.63)0.242.49 (0.99)2.24 (0.47)0.0420.23 (0.08) TABLE 2: GENE EXPRESSION PROFILE RESISTANT VS NON-RESISTANT AML Gene ΔΔ Ct (mean, SEM) 95% CI P-value Relative fold change JAK1 -4.63 (1.98) -9.48 0.21 0.0579 24.83 JAK2 -5.38 (0.94) -7.67 -3.08 0.0012 41.52 JAK3 -5.90 (2.17) -12.81 1.01 0.0726 59.77 S100A8 -7.16 (2.66) -14.01 -0.32 0.0432 143.27 S100A9 -8.31 (2.75) -15.04 -1.59 0.0233 318.37 c-myc -2.78 (0.59) -4.24 -1.33 0.0034 6.89 Refs: 1.Davis et al. Cancer Research 2010;70(13):5249 2.Jeyaraj A, Chen X, Szabolcs P. IL-15 Induced Polyclonal CTL Generated From Expanded CBT Cells Against Leukemia Cell Lines Constitutes IFN-γ Producing Cells and TCRγδ Cells. ASH 2012 Annual Meeting Figure 1 Figure 1. Figure 2 Figure 2. Figure 3 Figure 3. Disclosures No relevant conflicts of interest to declare.

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.

Ectopic Expression of Mer on Jurkat Cells and Its Anti-Apoptosis Effect.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 4098-4098
Author(s):  
Lei Fan ◽  
Liqian Xie ◽  
Fei Shen ◽  
Lan Dai ◽  
Jianyong Li ◽  
...  
Keyword(s):  
T Cells ◽  
Tyrosine Kinase ◽  
Caspase 3 ◽  
Ectopic Expression ◽  
Leukemia Cell ◽  
Annexin V ◽  
Jurkat Cells ◽  
Leukemia Cell Line ◽  
Serum Starvation ◽  
Significant Difference

Abstract BACKGROUND & OBJECTIVE: Mer is one member of Axl receptor tyrosine kinase family; its ligand Gas6 can bind Mer, then stimulate the tyrosine kinase activity and downstream cell signal pathway of Mer, and take part in cell inflammation, apoptosis and thrombosis. There are more and more reports on Mer function, while few on its association with malignant diseases. This study was to detect the expression of Mer on T-cell leukemia cell line--Jurkat, and investigate its anti-apoptosis function and the mechanism. METHODS: Flow cytometry (FCM) was used to detect Mer expression on normal T cells and Jurkat cells. RNA interference (RNAi) was used to block the expression of Mer on Jurkat cells. The effect of Mer on the proliferation of Jurkat cells was assessed by MTT assay, and its effect on serum starvation-induced apoptosis was evaluated by FCM with Annexin V/PI double staining. The expression of apoptosis-associated genes Bcl-2 and Caspase-3 in Jurkat cells was detected by real-time polymerase chain reaction (PCR) after Mer blocking. RUSELTS: Mer was not expressed in normal T cells both in peripheral blood and bone marrow, but highly expressed in Jurkat cells with a positive rate of 51.1%±5.2%. The inhibition rate of Mer expression in Jurkat cells by RNAi was 86.0%±10.3%. After 48-hour serum starvation, the apoptosis rate was 15.3%±4.3% in Mer-blocking Jurkat cells, and only 1.5%±1.0% in control Jurkat cells(P<0.01). There was no significant difference in Jurkat cell proliferation rate between these 2 groups. After Mer blocking, Bcl-2 expression was decreased by 42.7%±8.6% of control(P<0.01), Caspase-3 expression showed no significant change. CONCLUSION: Mer is highly expressed in Jurkat cells, and could inhibit cell apoptosis via Bcl-2 signaling pathway.

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