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

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 4122-4122
Author(s):  
Ikuo Murohashi ◽  
Noriko Ihara
Keyword(s):  
Stem Cells ◽  
Cell Lines ◽  
Leukemia Cell ◽  
Cell Number ◽  
Leukemia Cells ◽  
Lymphoid Leukemia ◽  
Hematopoietic Stem ◽  
Cultured Cell ◽  
Leukemia Cell Lines ◽  
Cell Densities

Abstract 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 leukemia cells has been shown to be stimulated by environmental niches (paracrine growth) or by cell-to-cell interaction and/or excreted factors of leukemia 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 of more than 105/ml) showed logarithmic linear increases in cell number, whereas those at lower cell densities (cultured cell densities of less than 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 Ab, which significantly decreased HL-60 leukemia cell growth. In contrast, anti-TNF-α Ab significantly stimulated the growth of the HL-60 cells. To clarify the nature of the cultured cell density on the growth of leukemia cells, leukemia cells were cultured at higher cell densities (group H, cultured cell densities of 106/ml) or at lower cell densities (group L, cultured cell densities of 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: Jagged-1, -1, Notch-1, -2, -3, Ang-1, -2, Tie-1, -2, Wnd3a, Wnd5a, β-Catenin, γ-Catenin, N-Cadherin, Cyclin D1, p16, p21, HOXA6, HOXA7, HOXA10, HOXB4, and Mef2c. At 24-hr cultures, transcripts of myeloid leukemia cell lines for Bmi-1, Wnt-3a, β-Catenin and γ-Catenin were higher, and those of lymphoid leukemia cell lines for Notch 1, 2, and 3 were higher in group H compared with group L. Transcript levels for Wnt5a were higher at 10-hr culture (HL-60 and Raji), those for HOXA7 at 30–10-hr (MO-7E, U-266 and Raji), and those for Mef2c at 3-hr (MO-7E, U-266 and Raji) in group H compared with group L. Taken together, our present results favor the conclusions that genes related to transcription factors and growth factors are sequentially and differentially expressed through cell-to-cell interaction of leukemia cells. The nature of the leukemia cell-to-cell interacrtion, which is related to the growth advantages of leukemia stem cells over normal hematopoietic stem cells, remains to be further clarified.

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.

Aurora-A kinase: a novel target of cellular immunotherapy for leukemia

Blood ◽  
2009 ◽  
Vol 113 (1) ◽  
pp. 66-74 ◽  
Author(s):  
Toshiki Ochi ◽  
Hiroshi Fujiwara ◽  
Koichiro Suemori ◽  
Taichi Azuma ◽  
Yoshihiro Yakushijin ◽  
...  
Keyword(s):  
Cell Lines ◽  
Leukemia Cell ◽  
Myelogenous Leukemia ◽  
Leukemia Cells ◽  
Cellular Immunotherapy ◽  
Aurora A Kinase ◽  
Aurora A ◽  
Hematopoietic Stem ◽  
Specific Ctls ◽  
Leukemia Cell Lines

Abstract Aurora-A kinase (Aur-A) is a member of the serine/threonine kinase family that regulates the cell division process, and has recently been implicated in tumorigenesis. In this study, we identified an antigenic 9–amino-acid epitope (Aur-A207-215: YLILEYAPL) derived from Aur-A capable of generating leukemia-reactive cytotoxic T lymphocytes (CTLs) in the context of HLA-A*0201. The synthetic peptide of this epitope appeared to be capable of binding to HLA-A*2402 as well as HLA-A*0201 molecules. Leukemia cell lines and freshly isolated leukemia cells, particularly chronic myelogenous leukemia (CML) cells, appeared to express Aur-A abundantly. Aur-A–specific CTLs were able to lyse human leukemia cell lines and freshly isolated leukemia cells, but not normal cells, in an HLA-A*0201–restricted manner. Importantly, Aur-A–specific CTLs were able to lyse CD34+ CML progenitor cells but did not show any cytotoxicity against normal CD34+ hematopoietic stem cells. The tetramer assay revealed that the Aur-A207-215 epitope–specific CTL precursors are present in peripheral blood of HLA-A*0201–positive and HLA-A*2402–positive patients with leukemia, but not in healthy individuals. Our results indicate that cellular immunotherapy targeting Aur-A is a promising strategy for treatment of leukemia.

The CXCR4 Antagonist AMD3100 Has Dual Effects, Initially Enhancing and Later Inhibiting Survival and Proliferation, in Myeloid Leukemia Cells in Vitro.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 1721-1721
Author(s):  
Ha-Yon Kim ◽  
Ji-Young Hwang ◽  
Seong-Woo Kim ◽  
Gak-Won Yun ◽  
Young-Joon Yang ◽  
...  
Keyword(s):  
Cell Lines ◽  
Myeloid Leukemia ◽  
Leukemia Cell ◽  
Leukemia Cells ◽  
Dependent Manner ◽  
Cxcr4 Antagonist ◽  
Hematopoietic Stem ◽  
Leukemia Cell Lines ◽  
Number Of Cells

Abstract Abstract 1721 Poster Board I-747 AMD3100, a small bicyclam antagonist for chemokine receptor CXCR4, induces the peripheral mobilization of hematopoietic stem cells. It also induces the segregation of leukemia cells in the bone marrow microenvironment, which should enhance the chemosensitivity of the cells. Based on these observations, AMD3100 is being considered for clinical use. However, AMD3100 activates G-protein coupled with CXCR4 and acts as a partial CXCR4 agonist. In this study, we explored whether AMD3100 affects the proliferation and survival of myeloid leukemia cells. As demonstrated previously, both AMD3100 and T140, another CXCR4 antagonist, markedly inhibited stromal cell-derived factor-1 (SDF-1)-induced chemotaxis and induced the internalization of CXCR4 in myeloid leukemia cell lines (U937, HL-60, MO7e, KG1a, and K562 cells) and CD34+ primary human acute myeloid leukemia (AML) cells. SDF-1 alone did not stimulate the proliferation of these leukemia cells, nor did it rescue the cells from apoptosis induced by serum deprivation. By contrast, AMD3100, but not T140, stimulated the proliferation of all five leukemia cell lines and primary AML cells in a dose-dependent manner in serum-free conditions for up to 5 days (∼ 2-fold increases at a concentration of 10-5M), which was abrogated by pretreating the cells with pertussis toxin. AMD3100 binds to CXCR7, another SDF-1 receptor, and all of the cells examined in this study expressed CXCR4 on the cell surface to some extent. The proliferation-enhancing effects of AMD3100 were not changed by knocking-down CXCR7 using the siRNA technique, whereas knocking-down CXCR4 significantly delayed the enhanced proliferation induced by AMD3100. Neither AMD3100 nor T140 induced the phosphorylation of Akt, Stat3, MAPK p44/p42, or MAPK p38, which are involved in SDF-1 signaling. In extended cultures of these cells for up to 14 days, AMD3100, but not T140, induced a marked decrease in the number of cells, compared to the control, after incubation for 5-7 days. Adding SDF-1 at the beginning and middle of the incubation did not affect the early increase or later decrease in the number of cells. AMD3100 reduced the apoptosis of these cells to a modest degree over the first 5-7 days and then markedly increased it. Consistent with the proliferation assay, AMD3100 increased the number of leukemia cell colonies during the early period of the assay, while it markedly decreased the number and size of the colonies in the later period of the assay. In conclusion, AMD3100 exerts dual effects, initially enhancing and subsequently inhibiting the survival and proliferation, in myeloid leukemia cells in vitro. Disclosures No relevant conflicts of interest to declare.

Bortezomib Interacts Synergistically with Belinostat to Induce Apoptosis In Human Acute Myeloid and Lymphoid Leukemia Cells

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 3266-3266
Author(s):  
Yun Dai ◽  
Shuang Chen ◽  
Li Wang ◽  
Xin-Yan Pei ◽  
Lora Kramer ◽  
...  
Keyword(s):  
Acute Leukemia ◽  
Cell Lines ◽  
Leukemia Cell ◽  
Dose Effect ◽  
Leukemia Cells ◽  
Parp Cleavage ◽  
Lymphoid Leukemia ◽  
Acute Leukemias ◽  
Leukemia Cell Lines ◽  
Acute Myeloid

Abstract Abstract 3266 Previous studies have demonstrated interactions between histone deacetylase (HDAC) and proteasome inhibitors (PIs) in multiple myeloma, NHL, and CLL. However, exploration of this strategy in acute leukemias has been more limited. In this context, we have previously demonstrated that HDACIs activate the cytoprotective NF-κB pathway in acute myeloid leukemia (AML) cells, and that interruption of this process dramatically increases lethality. Such findings raise the possibility that PIs, which block degradation of the NF-κB-inhibitory protein IκBα, may act via an analogous mechanism in acute leukemias. Consequently, interactions between the clinically relevant pan-HDAC inhibitor belinostat (PXD-101) and the FDA-approved proteasome inhibitor bortezomib were evaluated in both continuously cultured cell lines and primary AML and acute lymphoid leukemia (ALL) samples. First, whereas each agent individually displayed only modest toxicity, co-treatment for 24 hr or 48 hr with low concentrations of bortezomib (3 - 5 nM) and belinostat (50 - 300 nM) led to pronounced increases in apoptosis in diverse human acute leukemia cell lines (e.g., AML, U937, HL-60, MV-4-11/Flt3-ITD; T-cell ALL, Jurkat; B-cell ALL, SEM). Interactions between these agents were determined to be synergistic by Median Dose Effect analysis. Significantly, equivalent interactions were observed in multiple primary AML (n = 4) and ALL (n = 3) blast specimens, while largely sparing normal CD34+ hematopoietic cells isolated from umbilical cord blood (n = 4), as determined by annexin V/PI, DiOC6, and/or 7-AAD uptake by flow cytometry. Western blot analysis demonstrated that co-exposure of primary leukemia blasts to bortezomib and belinostat resulted in marked increase in PARP cleavage, compared with each agent administrated alone. In addition, cell morphology exhibited classical features of apoptosis in primary acute leukemia blasts, but not in normal CD34+ cells, following combination treatment. Second, in both cell lines and primary blasts, administration of bortezomib resulted in accumulation of the phosphorylated (S32/S36) form of IκBα, accompanied by diminished belinostat-mediated hyperacetylation (K310) of RelA/p65. Bortezomib also blocked processing of the precursor p100 into the active p52, an event enhanced by co-treatment with belinostat. These results indicate that a regimen combining bortezomib and belinostat interrupts both canonical and non-canonical NF-κB signaling pathways in acute leukemia cells. Moreover, co-exposure to these agents diminished expression of NF-κB-dependent pro-survival proteins including Bcl-xL, XIAP, and SOD2, but not NF-κB-independent anti-apoptotic proteins such as survivin. Third, because the BH3-only Bcl-2 family pro-apoptotic protein Bim plays an important role in the lethality of PIs or HDACIs as single agents, the expression and functional role of Bim in bortezomib/belinostat interactions was examined. Notably, whereas treatment with marginally toxic concentrations of either agent alone clearly increased Bim protein levels, co-exposure of either leukemia cell lines or primary blasts to bortezomib and belinostat led to sharply increased Bim expression (particularly the BimEL isoform). Importantly, shRNA knock-down of Bim substantially attenuated lethality mediated by co-treatment with bortezomib and belinostat in both AML (U937) and ALL (Jurkat) cells, supporting the notion that up-regulation of Bim plays a critical role in anti-leukemic activity of the combination regimen. Lastly, exposure of cultured leukemia cells and primary blasts to belinostat ± bortezomib induced hyperacetylation of a-tubulin, indicating inhibition of HDAC6, a microtubule-associated deacetylase that regulates aggresome formation and cell survival in response to misfolded protein-induced stress. Together, these findings indicate that the regimen combining belinostat and bortezomib is highly active against human AML and ALL cells, including primary leukemic blasts, in association with perturbation in the balance between pro-survival (NF-κB-dependent) and pro-death (e.g., Bim) signals. They also suggest that this strategy warrants further attention in acute leukemias. Accordingly, a Phase I trial of belinostat and bortezomib in patients with refractory acute leukemia or MDS has recently been initiated. Disclosures: Off Label Use: Investigational use of belinostat and bortezomib.

PD0332991, a CDK4/6 Inhibitor, Has An Anti-Leukemic Activity Against Lymphoid Crisis of CML and Ph+ALL Even with T315I Mutation in BCR-Abl; in Vitro Analysis

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 1511-1511
Author(s):  
Atsushi Nemoto ◽  
Takeshi Inukai ◽  
Koshi Akahane ◽  
Hiroko Honna- Oshiro ◽  
Kumiko Goi ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Line ◽  
Cell Lines ◽  
Cell Cycle Progression ◽  
Leukemia Cell ◽  
Leukemia Cells ◽  
Lymphoid Leukemia ◽  
Cycle Progression ◽  
Leukemia Cell Lines ◽  
T315i Mutation

Abstract Abstract 1511 Since BCR-ABL plays a central role in cell cycle progression of Philadelphia-chromosome positive (Ph+) leukemia cells and CDK4/6 critically involves in G1-progression of cell cycle, we analyzed sensitivity of Ph+ leukemia cell lines to compounds that act as specific CDK4/6 inhibitors. H3-thymidine uptake assay showed that both PD183812 and CBC219476 significantly inhibited cell growth of Ph+ lymphoid leukemia cell lines (n=9) in comparison with Ph+ myeloid leukemia cell lines (n=7) and Ph- ALL cell lines (n=26). Thus, we next tested the anti-leukemic activity of PD0332991, a potent CDK4/6 inhibitor that is under phase II clinical study for solid tumor patients, and found that 8 of 9 Ph+ lymphoid leukemia cell lines showed extremely higher sensitivity to PD0332991; median IC50 was <25 nM. IC50 of Ph+ lymphoid leukemia cell lines was significantly lower than that of Ph+ myeloid cell lines (200 nM, n=7) and Ph-ALL cell lines (100nM, n=25). PD0332991 effectively dephosphorylated Rb protein (pRb), and subsequently induced G1 arrest on all of Ph+ lymphoid leukemia cell lines. Moreover, PD0332991 gradually induced cell death in 4 Ph+ lymphoid leukemia cell lines. Since CDK4/6 inhibitor acts depending on intact pRb, we analyzed protein and gene expression status of Rb. Of note, all Ph+ lymphoid leukemia cell lines expressed intact pRb except for one cell line that showed relative resistance to PD0332991. In contrast, pRb was almost undetectable in Ph+ myeloid cell lines in spite of comparable level of Rb gene expression, which might be mechanism for resistance to PD0332991. However, most of Ph- ALL cell lines had intact pRb expression in spite of their relative resistance to PD0332991, indicating that Rb status alone did not explain higher PD0332991-sensitivity of Ph+ lymphoid leukemia cell lines. Thus, we assumed that Ph+ lymphoid leukemia cells showed higher PD0332991-sensitivity probably because BCR-ABL regulates CDK4/6 expression for cell cycle progression. To clarify this assumption, we treated Ph+ lymphoid leukemia cell lines with imatinib and performed immunoblot analysis of cell cycle machineries such as CDKs, cyclines, and CDK inhibitors. Of note, CDK4 expression level was frequently downregulated by imatinib in Ph+ lymphoid leukemia cell lines. Moreover, imatinib-induced downregulation of CDK4 in Ph+ lymphoid leukemia cell line was abrogated by the addition of IL-7 and FLT3 ligand, which stimulated cell cycle progression of imatinib-treated Ph+ ALL cell line. LY294002, a PI3K inhibitor, but not U0126, a MAPK inhibitor, and AG490, an inhibitor for JAK/STAT pathway, efficiently downregulated CDK4 expression in Ph+ lymphoid leukemia cell lines. Gene expression level of CDK4 in Ph+ lymphoid leukemia cell lines was downregulated by imatinib, and lactastatin, an inhibitor of protein degradation, partially inhibited imatinib-induced downregulation of CDK4 protein in Ph+ lymphoid leukemia cell lines, indicating that BCR-ABL regulates CDK4 expression both in gene expression level and in protein degradation level. These findings indicated that Ph+ lymphoid leukemia cell lines showed higher sensitivity to PD0332991 since BCR-ABL induces cell cycle progression of Ph+ lymphoid leukemia cells by regulating CDK4 as one of downstream pathways. Accordingly, we tested if PD0332991 shows anti-leukemic activity in Ph+ lymphoid leukemia cells that have a T315I mutation of BCR-ABL. SU/SR is an imatinib-resistant Ph+ ALL cell line with T315I mutation (IC50 for imatinib >10 mM), which was established from SU-Ph2, an imatinib-sensitive Ph+ ALL cell line (IC50 for imatinib <0.1 mM), after long-term culture in the presence of gradually increasing concentration of imatinib. Of note, PD0332991 effectively dephosphorylated pRb and inhibited cell growth of both SU/SR and SU-Ph2. Our findings provide a rationale for efficacy of PD0332991 in the context of anti-leukemic therapy for lymphoid crisis of CML and Ph+ ALL patients even with T315I mutation in BCR-ABL. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Ampelopsin Inhibits Cell Proliferation and Induces Apoptosis in HL60 and K562 Leukemia Cells by Downregulating AKT and NF-κB Signaling Pathways

2021 ◽  
Vol 22 (8) ◽  
pp. 4265
Author(s):  
Jang Mi Han ◽  
Hong Lae Kim ◽  
Hye Jin Jung
Keyword(s):  
Signaling Pathways ◽  
Cell Lines ◽  
White Blood Cells ◽  
Leukemia Cell ◽  
Leukemia Cells ◽  
Ros Generation ◽  
Anticancer Activities ◽  
Nuclear Condensation ◽  
Leukemia Cell Lines ◽  
Antileukemic Effect

Leukemia is a type of blood cancer caused by the rapid proliferation of abnormal white blood cells. Currently, several treatment options, including chemotherapy, radiation therapy, and bone marrow transplantation, are used to treat leukemia, but the morbidity and mortality rates of patients with leukemia are still high. Therefore, there is still a need to develop more selective and less toxic drugs for the effective treatment of leukemia. Ampelopsin, also known as dihydromyricetin, is a plant-derived flavonoid that possesses multiple pharmacological functions, including antibacterial, anti-inflammatory, antioxidative, antiangiogenic, and anticancer activities. However, the anticancer effect and mechanism of action of ampelopsin in leukemia remain unclear. In this study, we evaluated the antileukemic effect of ampelopsin against acute promyelocytic HL60 and chronic myelogenous K562 leukemia cells. Ampelopsin significantly inhibited the proliferation of both leukemia cell lines at concentrations that did not affect normal cell viability. Ampelopsin induced cell cycle arrest at the sub-G1 phase in HL60 cells but the S phase in K562 cells. In addition, ampelopsin regulated the expression of cyclins, cyclin-dependent kinases (CDKs), and CDK inhibitors differently in each leukemia cell. Ampelopsin also induced apoptosis in both leukemia cell lines through nuclear condensation, loss of mitochondrial membrane potential, increase in reactive oxygen species (ROS) generation, activation of caspase-9, caspase-3, and poly ADP-ribose polymerase (PARP), and regulation of Bcl-2 family members. Furthermore, the antileukemic effect of ampelopsin was associated with the downregulation of AKT and NF-κB signaling pathways. Moreover, ampelopsin suppressed the expression levels of leukemia stemness markers, such as Oct4, Sox2, CD44, and CD133. Taken together, our findings suggest that ampelopsin may be an attractive chemotherapeutic agent against leukemia.

scholarly journals A Novel, Myeloid Transcription Factor, C/EBPε, Is Upregulated During Granulocytic, But Not Monocytic, Differentiation

Blood ◽  
1997 ◽  
Vol 90 (7) ◽  
pp. 2591-2600 ◽  
Author(s):  
Roberta Morosetti ◽  
Dorothy J. Park ◽  
Alexey M. Chumakov ◽  
Isabelle Grillier ◽  
Masaaki Shiohara ◽  
...  
Keyword(s):  
Stem Cells ◽  
Transcription Factor ◽  
Hematopoietic Stem Cells ◽  
Cell Line ◽  
Cell Lines ◽  
Myeloid Leukemia ◽  
Leukemia Cell ◽  
Rt Pcr ◽  
Hematopoietic Stem ◽  
Nb4 Cells

Human C/EBPε is a newly cloned CCAAT/enhancer-binding transcription factor. Initial studies indicated it may be an important regulator of human myelopoiesis. To elucidate the range of expression of C/EBPε, we used reverse transcription-polymerase chain reaction (RT-PCR) analysis and examined its expression in 28 hematopoietic and 14 nonhematopoietic cell lines, 16 fresh myeloid leukemia samples, and normal human hematopoietic stem cells and their mature progeny. Prominent expression of C/EBPε mRNA occurred in the late myeloblastic and promyelocytic cell lines (NB4, HL60, GFD8), the myelomonoblastic cell lines (U937 and THP-1), the early myeloblast cell lines (ML1, KCL22, MDS92), and the T-cell lymphoblastic leukemia cell lines CEM and HSB-2. For the acute promyelocytic leukemia cell line NB4, C/EBPε was the only C/EBP family member that was easily detected by RT-PCR. No C/EBPε mRNA was found in erythroid, megakaryocyte, basophil, B lymphoid, or nonhematopoietic cell lines. Most acute myeloid leukemia samples (11 of 12) from patients expressed C/EBPε. Northern blot and RT-PCR analyses showed that C/EBPε mRNA decreased when the HL60 and KG-1 myeloblast cell lines were induced to differentiate toward macrophages. Similarly, Western blot analysis showed that expression of C/EBPε protein was either unchanged or decreased slightly as the promyelocytic cell line NB4 differentiated down the macrophage-like pathway after treatment with a potent vitamin D3 analog (KH1060). In contrast, C/EBPε protein levels increased dramatically as NB4 cells were induced to differentiate down the granulocytic pathway after exposure to 9-cis retinoic acid. Furthermore, very early, normal hematopoietic stem cells (CD34+/CD38−), purified from humans had very weak expression of C/EBPε mRNA, but levels increased as these cells differentiated towards granulocytes. Likewise, purified granulocytes appeared to express higher levels of C/EBPε mRNA than purified macrophages. Addition of phosphothiolated antisense, but not sense oligonucleotides to C/EBPε, decreased clonal growth of HL-60 and NB4 cells by about 50% compared with control cultures. Taken together, our results indicate that expression of C/EBPε is restricted to hematopoietic tissues, especially myeloid cells as they differentiate towards granulocytes and inhibition of its expression in HL-60 and NB4 myeloblasts and promyelocytes decreased their proliferative capacity. Therefore, this transcriptional factor may play an important role in the process of normal myeloid development.

Effect of (dl)-5-methyltetrahydrofolate on lymphoid leukemia cell lines

1991 ◽  
Vol 15 (7) ◽  
pp. 645-649 ◽  
Author(s):  
Pietro Antonio Bernabei ◽  
William I. Bensinger
Keyword(s):  
Cell Lines ◽  
Leukemia Cell ◽  
Lymphoid Leukemia ◽  
Leukemia Cell Lines

Metabolic Profile Of Leukemia Cells Influences Treatment Efficacy Of L‑Asparaginase

2019 ◽  
Author(s):  
Katerina Hlozkova ◽  
Alena Pecinova ◽  
David Pajuelo Reguera ◽  
Marketa Simcikova ◽  
Lenka Hovorkova ◽  
...  
Keyword(s):  
Membrane Potential ◽  
Mitochondrial Membrane Potential ◽  
Cell Lines ◽  
Mitochondrial Membrane ◽  
Metabolic Profile ◽  
Lymphoblastic Leukemia ◽  
Leukemia Cell ◽  
Leukemia Cells ◽  
Lower Sensitivity ◽  
Leukemia Cell Lines

Abstract Background Effectiveness of L-asparaginase administration in acute lymphoblastic leukemia treatment is mirrored in overall outcome of patients. Generally, leukemia patients differ in their sensitivity to L-asparaginase; however, the mechanism underlying their inter-individual differences is still not fully understood. We have previously shown that L-asparaginase rewires the biosynthetic and bioenergetic pathways of leukemia cells to activate both anti-leukemic and pro-survival processes. Herein, we investigated the relationship between the metabolic profile of leukemia cells and their sensitivity to currently used cytostatic drugs.Methods Altogether, 19 leukemia cell lines and primary leukemia cells from 11 patients were used. Glycolytic function and mitochondrial respiration were measured using Seahorse bioanalyzer. Sensitivity to cytostatics was measured using MTS assay and/or absolute count and flow cytometry. Mitochondrial membrane potential was determined as TMRE fluorescence.Results We characterized the basal metabolic state of the cells derived from different leukemia subtypes using cell lines and primary samples and assessed their sensitivity to cytostatic drugs. We found that leukemia cells cluster into distinct groups according to their metabolic profile, which is mainly driven by their hematopoietic lineage of origin from which they derived. However, majority of lymphoid leukemia cell lines and patients with lower sensitivity to L-asparaginase clustered regardless their hematopoietic phenotype together with myeloid leukemias. Furthermore, we observed a correlation of specific metabolic parameters with sensitivity to L-asparaginase. Greater ATP-linked respiration and lower basal mitochondrial membrane potential in cells significantly correlated with higher sensitivity to L-asparaginase. No such correlation was found in other tested cytostatic drugs.Conclusions These data support the prominent role of the cell metabolism in the treatment effect of L-asparaginase. Based on these findings metabolic profile could identify leukemia patients with lower sensitivity to L-asparaginase with no specific genetic characterization.

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