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.

Analysis of Aurora Kinase Expressions and Cell Cycle Regulation by Aurora-C in Leukemia Cells.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1366-1366 ◽  
Author(s):  
Miki Kobayashi ◽  
Satoki Nakamura ◽  
Takaaki Ono ◽  
Yuya Sugimoto ◽  
Naohi Sahara ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Western Blot ◽  
Cell Lines ◽  
Mammalian Cells ◽  
Cell Cycle Progression ◽  
Leukemia Cell ◽  
Leukemia Cells ◽  
Human Leukemia ◽  
Aurora A ◽  
Aurora Kinases

Abstract Background: The conserved Aurora family kinases, a family of mitotic serine/threonine kinases, have three members (Aurora-A, -B and -C) in mammalian cells. The Aurora kinases are involved in the regulation of cell cycle progression, and alterations in their expression have been shown to associate with cell malignant transformation. Aurora A localizes to the centrosomes during anaphase, and it is required for mitotic entry. Aurora B regulates the formation of a stable bipolar spindle-kinetochore attachment in mitosis. The function of Aurora-C in mammalian cells has not been studied extensively. In this study, we investigated that human leukemia cells expressed all 3 Aurora kinases at both protein and mRNA level, and the mechanisms of cell cycle regulation by knock down of Aurora C in leukemia cells. Methods: In this study, we used the 7 human leukemia cell lines, K562, NB4, HL60, U937, CEM, MOLT4, SUP-B15 cells. The expression levels of mRNA and proteins of Aurora kinases were evaluated by RT-PCR and western blot. The analysis of proliferation and cell cycle were performed by MTT assay and FCM, respectively. Results: The mRNA of Aurora-A and Aurora-B are highly expressed in human leukemia cell lines (K562, NB4, HL60, U937, CEM, MOLT4, SUP-B15 cells), while the mRNA of Aurora C is not only expressed highly in all cells. In contrast, an increase in the protein level of the 3 kinases was found in all cell lines. These observations suggested posttranscriptional mechanisms, which modulate the expression of Aurora C. In cell cycle analysis by flow cytometory, the knock down of Aurora C by siRNA induced G0/G1 arrest and apoptosis in leukemia cells, and increased the protein levels of p27Kip1 and decreased Skp2 by western blot. In MTT assay, it was revealed that the growth inhibition of leukemia cells transfected with siRNA Aurora C compared with leukemia cells untransfected with siRNA Aurora C. Moreover, We showed that Aurora C was associated with Survivin and directly bound to Survivin by immunoprecipitation and western blot. Conclusion: We found that human leukemia cells expressed all 3 members of the Aurora kinase family. These results suggest that the Aurora kinases may play a relevant role in leukemia cells. Among these Aurora kinases, Aurora C interacted with Survivin and prevented apoptosis of leukemia cells, and induced cell cycle progression. Our results showed that Aurora-C may serve as a key regulator in cell division and survival. These results suggest that the Aurora C kinase may play an important role in leukemia cells, and may represent a target for leukemia therapy.

Gene Profiling Mechanisms of Cell Density-Dependent Growth in Myeloid and Lymphoid Leukemia Cell Lines.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 4420-4420
Author(s):  
Ikuo Murohashi ◽  
Noriko Ihara
Keyword(s):  
Gene Expression ◽  
Growth Factors ◽  
Cell Density ◽  
Cell Lines ◽  
Leukemia Cell ◽  
Leukemic Cells ◽  
Leukemia Cells ◽  
Lymphoid Leukemia ◽  
Cultured Cell ◽  
Cell Densities

Abstract Abstract 4420 Normal hematopoietic stem cells have been shown to be maintained through interaction with their environmental niches, such as osteoblastic and endothelial ones. The growth of leukemic cells has been shown to be stimulated by environmental niches (paracrine growth) or by cell-to-cell interaction or excreted factors of leukemic cells (autocrine growth). The growth of myeloid (MO7-E and HL-60) and lymphoid (Raji, U-266, Daudi and RPMI-1788) leukemia cell lines cultured at various cell densities in serum free medium (Sigma H 4281) with 1% BSA was evaluated. The cells cultured at higher cell densities (cultured cell densities ≥a 105/ml) showed logarithmic linear increases in cell number, whereas those at lower cell densities (cultured cell densities □… 104/ml) ceased increasing cell number. Supernatants of myeloid leukemia cells stimulated the growth of autologous clonogenic cells, but not those of lymphoid leukemia cells. Neutralizing antibodies (Abs) against various hematopoietic growth factors failed to inhibit cell growth except for anti-VEGF, which significantly decreased HL-60 leukemia cell growth. To clarify the nature of the cultured cell density on the growth of leukemia cells, leukemia cells were cultured at higher cell density (group H, cultured cell densities of 106/ml) or at lower cell density (group L, cultured cell densities 104/ml). After culture of 3-, 6-, 10-, and 24-hr, cells were serially harvested and total cellular RNA was extracted. Gene transcript levels were determined by using Real-Time PCR. Gene transcripts examined in the present study were as follows: polycomb (Bmi1), Hox (HOXA7, HOXA9, HOXB2, HOXB4, Meis 1), Caudal-related (CDX2, 4), Mef2c, c-Myb, Wnt (Wnt 3a, Wnt 5a, β-Catenin, β-Catenin, N-Cadherin), Notch (Notch-1, -2, -3 and Jagged-1, -2), CKI (p14, p15, p16, p18, p21, p27, p57), growth factor (VEGF, IGF-1, -2, Ang-1, -2, SDF-1), growth factor receptor (Flt-1, KDR, neurophilin-1, IGF-1R, Tie-1, -2, CXCR4), and growth related (c-Myc, CyclinD1, Foxo3a) genes. p18 and p21 gene expression was higher in group L compared with group H in two and all five groups, respectively. In contrast, p14 gene expression was higher in group H compared with group L. Any of the p15, p16, p27 and p57 genes was deleted. VEGF gene expression levels at 1-3- hr culture were higher in group H compared with group L. HOX, Meis 1 and Mef2c gene expression levels at 1- to 10- hr culture were higher in group H compared with group L. At 24-hr cultures, transcripts of myeloid and lymphoid cell lines for Bmi-1, Wnt-3a, and β-Catenin were higher, and those of lymphoid cell lines for Notch 1, 2, and 3 were higher in group H compared with group L. Taken together, our present results favor the conclusions that genes related to growth factors and transcription factors are sequentially and differentially expressed through cell-to-cell interaction and excreted autocrine growth factors of leukemia cells. Disclosures: No relevant conflicts of interest to declare.

Dysregulated Expression of HSP70 Isoforms in Acute Myelogenous Leukemia (AML)

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 1370-1370
Author(s):  
Krishan K. Sharma ◽  
Juan Felipe Rico ◽  
Duane C Hassane ◽  
Gabriela Chiosis ◽  
Monica L. Guzman
Keyword(s):  
Drug Resistance ◽  
Heat Shock ◽  
Cord Blood ◽  
Cell Lines ◽  
Blood Cells ◽  
Leukemia Cell ◽  
Leukemia Cells ◽  
Normal Cells ◽  
Leukemia Cell Lines ◽  
Cord Blood Cells

Abstract Abstract 1370 Stress-inducible heat shock protein 70 (HSP70) is a major cytoprotective factor and a molecular chaperone that interacts with HSP90 to form a multi-chaperone complex. Cancer cells are highly dependent on this complex due to their increased demand for protein synthesis. HSP70 overexpression inhibits apoptosis and has been associated with drug resistance. However, the contribution to drug resistance in AML of specific HSP70 isoforms remains unknown. As there is growing interest in therapeutically targeting HSP70, we investigated the expression of 7 different HSP70 isoforms in AML primary cells and leukemia cell lines and their response to a novel HSP70-inhbitor, YK5. A panel of 12 leukemia cell lines and 11 primary samples was used to determine the expression of HSP70 and their response to YK5. We also evaluated the changes to the HSP70 isoforms when exposed to either heat shock or YK5. We found MV4-11, MOLM-13, and U937 sensitive to YK5 (LD50 = 1.18μM, 1.03μM, and 2.31μM at 24 hours, respectively). In contrast, OCI-AML3, TUR and THP-1 were more resistant to the inhibitor. (LD50 = 9.92μM, 9.74μM, and 8.84μM at 24 hours, respectively). Non-tumor cells, however, were significantly less affected by treatment with YK5 (72% viable cord blood mononuclear cells after 24 hour treatment with 5μM YK5). We found that the cell surface expression of HSP70 was higher in both cell lines and primary samples when compared their normal counterparts. Furthermore, quantitative PCR revealed that cell lines with higher levels of HSPA1A and lower levels of HSPA6 demonstrated higher sensitivity to YK5. Interestingly, higher levels of HSPA1A and lower levels of HSPA6 were also found in primary AML samples when compared to CD34+ cord blood cells, consistent with the relative insensitivity of normal cells to YK5. We further discovered, mining publicly available databases, that high levels of HSPA1A were associated poorer prognosis (p = 0.004), suggesting that YK5 would be beneficial to patients presenting high HSP70 expression. We also evaluated the effect of YK5 on the gene expression of the various HSP70 isoforms. Quantitative PCR revealed the ability of YK5 to downregulate HSPA6 and HSC70 (HSPA8) in both cell lines and primary samples. Strikingly, all HSP70 isoforms exhibited similar fold changes upon heat shock in primary samples, CD34+ cord blood cells, and leukemia cell lines, indicating that the cellular stress response is not damaged in AML. However, the specificity of HSP70 inhibition to leukemia cells and not normal cells suggests a dysregulated set of client proteins and increased dependency on HSP70 to maintain leukemic homeostasis. In summary, we have found dysregulated expression of the HSP70 isoforms HSPA1A and HSPA6 in leukemia cells and that the expression levels of these isoforms correlate to the sensitivity of YK5-mediated HSP70 inhibition (HSPA1A: p=0.0012 and r2=0.801, HSPA6: p=0.0011 and r2=0.847). *KKS and JFR contributed equally to this project Disclosures: No relevant conflicts of interest to declare.

RASSF2 Functions As a Tumor Suppressor in t(8;21) AML Via Promotion of Apoptosis

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 3590-3590
Author(s):  
Samuel A Stoner ◽  
Russell Dekelver ◽  
Miao-Chia Lo ◽  
Dong-Er Zhang
Keyword(s):  
Gene Expression ◽  
Tumor Suppressor ◽  
Cell Lines ◽  
Hematopoietic Cells ◽  
Leukemia Cell ◽  
Leukemia Cells ◽  
Mrna Levels ◽  
Human Cd34 ◽  
Leukemia Cell Lines ◽  
Cd34 Cells

Abstract The t(8;21) chromosomal translocation is one of the most common chromosomal translocations associated with acute myeloid leukemia (AML), found in approximately 12% of de novo AML cases. The majority of these cases are classified as FAB-subtype M2 AML. The t(8;21) results in the stable fusion of the AML1 (RUNX1) and ETO (RUNX1T1) genes. The AML1-ETO fusion protein is composed of the N-terminal portion of AML1, which includes the DNA-binding Runt-homology domain, and nearly the full-length ETO protein. The primary accepted mechanism by which AML1-ETO promotes leukemia development is through the aberrant recruitment of transcriptional repression/activation complexes to normal AML1 target genes. Therefore, the identification of individual genes or biological pathways that are specifically disrupted in the presence of AML1-ETO will provide further molecular insight into the pathogenesis of t(8;21) AML and lead to the possibility for improved treatment for these patients. We identified RASSF2 as a gene that is specifically downregulated in (2-4 fold) in total bone marrow of t(8;21) patients compared to non-t(8;21) FAB-subtype M2 AML patients by analyzing publicly available gene expression datasets. Similarly, using a mouse model of t(8;21) AML we found Rassf2 mRNA levels to be nearly 30-fold lower in t(8;21) leukemia cells compared to wild-type Lin-Sca-cKit+ (LK) myeloid progenitors. Gene expression analysis by RT-qPCR in leukemia cell lines confirmed that RASSF2 mRNA levels are significantly downregulated (8-10-fold) in both Kasumi-1 and SKNO-1 t(8;21) cell lines as compared to a similar non-t(8;21) HL-60 cell line and to primary human CD34+ control cells. In addition, expression of AML1-ETO in HL-60 or CD34+ cells results in a decrease in RASSF2 mRNA expression, which further suggests that RASSF2 is a target for regulation by AML1-ETO. Assessment of published ChIP-seq data shows that AML1-ETO binds the RASSF2 gene locus at two distinct regions in both primary t(8;21) AML patient samples and in the Kasumi-1 and SKNO-1 cell lines. These regions are similarly bound by several important hematopoietic transcription factors in primary human CD34+ cells, including AML1, ERG, FLI1, and TCF7L2, implicating these two regions as important for the regulation of RASSF2 expression during blood cell differentiation. Overexpression of RASSF2 in human leukemia cell lines using an MSCV-IRES-GFP (MIG) construct revealed that RASSF2 has a strong negative effect on leukemia cell proliferation and viability. The overall percentage of GFP-positive cells in MIG-RASSF2 transduced cells markedly decreased compared to MIG-control transduced cells over a period of 14 days. This effect was primarily due to significantly increased apoptosis in the RASSF2 expressing cell populations. Similarly, we found that expression of RASSF2 significantly inhibits the long-term self-renewal capability of hematopoietic cells transduced with AML1-ETO in a serial replating/colony formation assay. AML1-ETO transduced hematopoietic cells were normally capable of serial replating for more than 6 weeks. However, AML1-ETO transduced cells co-expressing RASSF2 consistently had reduced colony number and lost their ability to replate after 3-4 weeks. This was due to a dramatically increased rate of apoptosis in RASSF2 expressing cells. RASSF2 is reported to be a tumor suppressor that is frequently downregulated at the transcriptional level by hypermethylation in primary tumor samples, but not healthy controls. Here we have identified RASSF2 as a target for repression, and demonstrated its tumor suppressive function in t(8;21) leukemia cells. Further insights into the molecular mechanisms of RASSF2 function in AML will continue to be explored. Disclosures No relevant conflicts of interest to declare.

JQ1, a Potential Therapeutic Molecule for Myeloid Leukemia with PTEN Deficiency

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 5899-5899 ◽  
Author(s):  
Nicholas J Baltz ◽  
Natalia C Colorado ◽  
Yan Yan ◽  
Shelly Lensing ◽  
Delli Robinson ◽  
...  
Keyword(s):  
Gene Expression ◽  
Western Blot ◽  
Colony Formation ◽  
Myeloid Leukemia ◽  
Leukemia Cell ◽  
K562 Cells ◽  
Leukemia Cells ◽  
Jak Inhibitors ◽  
Facs Analysis ◽  
Leukemia Cell Lines

Abstract Acute myeloid leukemia (AML) is a hematologic malignancy that continues to have high relapse and treatment-related mortality rates, despite recent advances in clinical management and therapy. Janus kinase (JAK) inhibitors inhibit the activity of the JAK/STAT pathway and have demonstrated some clinical responses in AML patients. However, survival analysis suggests that more than half of AML patients do not benefit from treatment with JAK inhibitors. Furthermore, PTEN deficiency is frequently found in patients in the late stages of cancer, which causes hyperactivated AKT and MAPK pathways. However, emerging data suggests that leukemia cells with PTEN deficiency are resistant to MAPK inhibitors. Over the past decade, it has been demonstrated that dysregulated epigenetics play an important role in myeloid leukemogenesis. The bromodomain and extraterminal domain (BET) family includes adaptor proteins Brd2, Brd3, Brd4, and Brdt that regulate gene expression via binding to acetylated chromatin and subsequently activating RNA Polymerase II driven transcriptional elongation, resulting in the promotion of gene expression. BRD4 is a BET protein required for disease maintenance in AML. JQ1 is a small molecule that interferes with transcriptional regulators, such as BRD4, by preventing them from interacting with acetylated regions of the genome and thus inhibiting the transcriptional activation of BRD4 target genes. Prior research in lymphocytic leukemia cell lines suggests that JQ1 also decreases STAT5-dependent gene transcriptional activities. We hypothesize that the inhibition of BET proteins may correct the over-activated transcriptional activities in myeloid leukemia cells and induce disease regression. We tested our hypothesis in PTEN deficient myeloid leukemia cell lines, TF-1a and K562, and used human cord blood mononuclear cells (CB) for normal cell comparison. Methods: 1) To test whether JQ1 can inhibit colony formation, we seeded cells on 0.3% agar and McCoys' 5A medium supplemented with nutrients and 15% fetal bovine serum, without cytokines, and added JQ1 diluents to the cultures at concentrations of 32.5-1000nM overnight after the cultures were established. 2) To test whether JQ1 can inhibit leukemia cell proliferation, we cultured cells in liquid medium with JQ1 for 48-72 hours, and quantified the viable cells using alamarBlue® assay. 3) To investigate whether JAK/STAT5 activity is altered by JQ1 in leukemia cells, we quantified phosphorylated STAT5 (pSTAT5) in cells via flow cytometry and western blot. We treated the cells with JQ1 at various concentrations for 2 hours and then stimulated the cells for 15 minutes in medium with 0.5% BSA and 10ng/mL GM-CSF prior to staining the cells with anti-pStat5 (pY694) antibody conjugated with Alexa Fluor® 647 for FACS analysis or lysing the cells for western blot analysis. Results: In the colony formation assay, we found that TF-1a cells were more sensitive to JQ1 than the CB cells and K562, with an IC50 of 62.5-125 nM for TF-1a cells (p<0.0001), and 250-500nM for both CB and K562 cells, respectively. Proliferation assay results also supported that TF1a cells are sensitive to JQ1 with an IC50 of 125-250nM, whereas neither CB nor K562 reached the IC50 in the tested concentration range. This suggests that the IC50 of JQ1 for TF1a cells is achievable at concentrations that are mostly nontoxic to normal CB cells, but K562 cells are not sensitive to JQ1. FACS analysis revealed that pSTAT5 is constitutively activated in K562 cells but not in TF-1a cells. Interestingly, the levels of pSTAT5 in both TF-1a and K562 cells were not altered by JQ1 treatment at tested concentrations, which was confirmed by western blot. Conclusions: Our data suggest: 1) JQ1 and other bromodomain inhibitors could be potential therapeutic molecules for selected myeloid leukemias; 2) JQ1 inhibition on colony formation and proliferation in TF-1a cells is not pSTAT5 related. Further studies are underway to test whether JQ1 is effective in primary mouse leukemia cells with Pten deficiency. Disclosures No relevant conflicts of interest to declare.

scholarly journals Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand (TRAIL) Mediates and Sustains NF-κb Constitutive Activation in LGL Leukemia Cells

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 2762-2762
Author(s):  
Jun Yang ◽  
Francis R Leblanc ◽  
Shubha Dighe ◽  
Susan B. Nyland ◽  
David J. Feith ◽  
...  
Keyword(s):  
Western Blot ◽  
Cell Lines ◽  
Leukemia Cell ◽  
Binding Activity ◽  
Leukemia Cells ◽  
Trail Expression ◽  
Constitutive Activation ◽  
Dna Binding Activity ◽  
Leukemia Cell Lines ◽  
Lgl Leukemia

Abstract Large granular lymphocyte (LGL) leukemia results from clonal expansion of CD3+ cytotoxic T-lymphocytes (CTL) or CD3-natural killer (NK) cells. Chronic antigen stimulation promotes long-term survival of LGL leukemia cells through constitutive activation of multiple survival pathways, leading to global dysregulation of apoptosis. Clinical manifestations of LGL leukemia include neutropenia, anemia and rheumatoid arthritis. Treatment for LGL leukemia patients relies on immunosuppressives such as methotrexate and is not curative. No standard therapy has been established. We reported that nuclear factor kappa B (NF-kB) is central to the leukemic LGL survival network, but the mechanisms of constitutive NF-kB activation in LGL leukemia are undefined. TNF-related apoptosis-inducing ligand (TRAIL) is a potent inducer of apoptosis and activates NF-kB via binding to TRAIL receptor (TR) 1, 2 and decoy receptor 2 (DcR2). DcR2 is uniquely unable to transduce downstream death signals yet retains NF-kB transduction activity. The mechanisms of TRAIL expression and regulation in LGL leukemia are unknown. Thus the current study investigates these mechanisms and their potential therapeutic applications, with the use of NF-kB inhibitors ixazomib and bortezomib, in LGL leukemia. Methods: LGL leukemia cell lines TL1 (T-LGL) and NKL (NK-LGL), peripheral blood mononuclear cells (PBMC) from LGL leukemia patients, and PBMC from normal donors were studied. NF-kB DNA binding activity was determined by EMSA. Results were confirmed using probe-based NF-kB (p50/65) transcription factor assay and immunocytochemistry (ICC). Serum TRAIL levels were detected by ELISA. Cellular TRAIL expression was determined by real-time PCR, western blot and ICC. DcR2 and Mcl-1 siRNA knock-down was performed with electroporation. Flow cytometry was used to detect TR 1-3 and DcR2 expression and apoptosis. Results: The average serum levels of TRAIL in LGL leukemia patients were nearly 4-fold higher than normal (NL) control values (p ≤ 0.0001). Data from RT-PCR (p ≤ 0.04), western blot and ICC revealed that LGL leukemia cells were the major source of TRAIL overexpression. Identical expression levels of TR1, 2 and 3 were observed in PBMC from LGL leukemia patients and from NL controls. Like normal PBMC, LGL leukemia cells were resistant to TRAIL-induced apoptosis. In contrast, the expression frequency of DcR2 was at least 4-fold greater in LGL leukemia PBMC compared to NL control, and it correlated to the percentage of circulating LGL leukemia cells. We found that TRAIL activates NF-kB and NF-kB downstream target genes, including TRAIL and McL-1, in LGL leukemia samples. To confirm that TRAIL is responsible for constitutive NF-kB activation in LGL leukemia, T-LGL leukemia PBMC were treated with pooled sera from 3 each of either NL controls or T-LGL leukemia patients. Leukemia sera increased NF-kB activity on EMSA, and this effect was completely blocked by TRAIL neutralizing antibody. DcR2 siRNA knockdown specifically decreased RelA and NF-kB1 (p105/p50) levels in TL1 and NKL cells. Mcl-1 siRNA mediated increased apoptosis in the same cell lines. Likewise, ixazomib and bortezomib facilitated leukemia-selective apoptosis in LGL leukemia cell lines and in patient PBMC, via inhibition of NF-kB activity and of downstream targets (ixazomib, p≤0.0001; bortezomib, p≤0.03). Additionally, caspase-3 and PARP cleavage were observed in LGL leukemia cells treated with ixazomib or bortezomib. Serum TRAIL levels in LGL leukemia patients were significantly lower in methotrexate responders versus non-responders, corresponding with reduced NF-kB DNA binding activity and increased absolute neutrophil counts, indicative of treatment response. Conclusion: These data indicate that expression of DcR2 and constitutive activation of NF-kB are responsible for TRAIL resistance in leukemic LGLs. TRAIL triggers prolonged NF-kB activation via interaction with DcR2, and activated NF-kB in turn promotes further TRAIL production in leukemic LGLs, creating a TRAIL autocrine regulatory loop. Inhibition of NF-kB activity with ixazomib and bortezomib interrupts this loop, impairs expression of Mcl-1 and induces apoptosis of leukemia cells. Our preclinical findings provide a solid framework for clinical evaluations of ixazomib and bortezomib in the treatment of LGL leukemia. Disclosures No relevant conflicts of interest to declare.

Anti-Apoptotic Proteins, HSP90 and Activated STAT3 Contribute to Busulfan Resistance of Myeloid Leukemia Cells.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 3472-3472
Author(s):  
Borje S. Andersson ◽  
Ben C. Valdez ◽  
David Murray ◽  
Latha Ramdas ◽  
Marcos de Lima ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Lines ◽  
Leukemia Cell ◽  
Myelogenous Leukemia ◽  
Leukemia Cells ◽  
Treatment Modalities ◽  
High Dose ◽  
Hematopoietic Stem ◽  
Conditioning Treatment ◽  
Apoptotic Genes

Abstract Busulfan(Bu)-based chemotherapy is a conditioning treatment prior to hematopoietic stem cell transplantation (HSCT) of patients with acute and chronic myelogenous leukemia (AML, CML). A major obstacle to successful HSCT is Bu resistance, which might be attributed to individual differences in drug pharmacokinetics and metabolism, or inherent resistance of cancer cells. We hypothesize that gene expression profiling of leukemia cells can be used to dissect the factors that contribute to their Bu resistance. Two Bu-resistant leukemia cell lines were established, characterized and analyzed by microarray and real-time RT-PCR techniques to identify differentially expressed genes. The CML B5/Bu2506 cells are 4.5-fold more resistant to Bu than their parental B5 cells. The AML KBM3/Bu2506 cells are 4.0-fold more Bu-resistant than KBM3 parental cells. Both resistant sublines evade Bu-mediated G2-arrest and apoptosis with constitutively up-regulated anti-apoptotic genes (BCL-XL, BCL2, BCL2L10, BAG3 and IAP2/BIRC3) and down-regulated pro-apoptotic genes (BIK, BNIP3, and LTBR).). Bu-induced apoptosis is partly mediated by activation of caspases; use of the inhibitor Z-VAD-FMK completely abrogated PARP1 cleavage and reduced apoptosis by ∼ 50%. Furthermore, Bu resistance in these cells may be attributed in part to up-regulation of HSP90 protein and activation of STAT3. Inhibition of HSP90 with geldanamycin attenuated phosphorylated STAT3 and made B5/Bu2506 and KBM3/Bu2506 more Bu-sensitive. Analysis of cells derived from patients classified as either clinically resistant or sensitive to high-dose Bu-based chemotherapy had alterations in gene expression that were analogous to those observed in the in-vitro model cell lines, confirming the potential clinical relevance of this model for Bu resistance. Our results suggest the important roles of apoptotic signaling mechanism, HSP90 and STAT3 and should be considered in the classification of AML patients who will likely benefit from busulfan-based pretransplant conditioning therapy and those who should be offered alternative treatment modalities.

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 Knowledge-guided gene prioritization reveals new insights into the mechanisms of chemoresistance

2016 ◽  
Author(s):  
Amin Emad ◽  
Carl R. Woese ◽  
Junmei Cairns ◽  
Krishna R. Kalari ◽  
Liewei Wang, M.D. ◽  
...  
Keyword(s):  
Gene Expression ◽  
Drug Resistance ◽  
Cell Lines ◽  
Gene Expression Data ◽  
Drug Response ◽  
Global Analysis ◽  
Extensive Literature ◽  
Expression Data ◽  
Chemotherapy Drugs ◽  
New Genes

ABSTRACTBackgroundIdentification of genes whose basal mRNA expression predicts the sensitivity of tumor cells to cytotoxic treatments can play an important role in individualized cancer medicine. It enables detailed characterization of the mechanism of action of drugs. Furthermore, screening the expression of these genes in the tumor tissue may suggest the best course of chemotherapy or a combination of drugs to overcome drug resistance.ResultsWe developed a computational method called ProGENI to identify genes most associated with the variation of drug response across different individuals, based on gene expression data. In contrast to existing methods, ProGENI also utilizes prior knowledge of protein-protein and genetic interactions, using random walk techniques. Analysis of two relatively new and large datasets including gene expression data on hundreds of cell lines and their cytotoxic responses to a large compendium of drugs reveals a significant improvement in prediction of drug sensitivity using genes identified by ProGENI compared to other methods. Our siRNA knockdown experiments on ProGENI-identified genes confirmed the role of many new genes in sensitivity to three chemotherapy drugs: cisplatin, docetaxel and doxorubicin. Based on such experiments and extensive literature survey, we demonstrate that about 73% our top predicted genes modulate drug response in selected cancer cell lines. In addition, global analysis of genes associated with groups of drugs uncovered pathways of cytotoxic response shared by each group.ConclusionsOur results suggest that knowledge-guided prioritization of genes using ProGENI gives new insight into mechanisms of drug resistance and identifies genes that may be targeted to overcome this phenomenon.

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.

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