Corinna Meyer

Activation of miR-17-92 by NK-like homeodomain proteins suppresses apoptosis via reduction of E2F1 in T-cell acute lymphoblastic leukemia

The NK-like family of homeobox genes includes TLX1, TLX3 and NKX2-5, which are ectopically activated in distinct subsets of T-cell acute lymphoblastic leukemia (T-ALL) cells. Here we analysed their effect on the miR-17-92 cluster overexpressed in several types of cancer, including T-ALL. The pri-miR-17-92 polycistron encodes micro-RNAs (miRNAs), which decrease E2F1 protein expression, regulating proliferation and/or apoptosis. Quantification of pri-miR-17-92 in T-ALL cell lines suggested an implication of the NK-like homeodomain proteins in transcriptional regulation. Lentiviral-mediated overexpression of NKX2-5 in the T-ALL cell line MOLT-4 consistently resulted in increased miR-17-92 pri-miRNA levels and decreased amounts of E2F1 protein. Induction of apoptosis by treating miR17-92 or E2F1 transduced T-ALL cells with etoposide led to reduced or enhanced cell viability, respectively. Furthermore, analysis of pri-miR-17-92 in T-ALL patients indicated elevated expression in those bearing TLX1/3 positive cells. These data support an activatory effect of NK-like homeodomain proteins on pri-miR-17-92 expression and concomitantly reduced E2F1 protein levels, thereby enhancing survival of leukemic T-cells.

Amplification at 7q22 targets cyclin-dependent kinase 6 in T-cell lymphoma

Recurrent chromosomal aberrations in hematopoietic tumors target genes involved in pathogenesis. Their identification and functional characterization are therefore important for the establishment of rational therapies. Here, we investigated genomic amplification at 7q22 in the T-cell lymphoma cell line SU-DHL-1 belonging to the subtype of anaplastic large-cell lymphoma (ALCL). Cytogenetic analysis mapped this amplicon to 86–95 Mb. Copy-number determination quantified the amplification level at 5- to 6-fold. Expression analysis of genes located within this region identified cyclin-dependent kinase 6 (CDK6) as a potential amplification target. In comparison with control cell lines, SU-DHL-1 expressed considerably higher levels of CDK6. Functionally, SU-DHL-1 cells exhibited reduced sensitivity to rapamycin treatment, as indicated by cell growth and cell cycle analysis. Rapamycin reportedly inhibits degradation of the CDK inhibitor p27 with concomitant downregulation of cyclin D3, implying a proliferative advantage for CDK6 overexpression. Amplification of the CDK6 locus was analyzed in primary T-cell lymphoma samples and, while detected infrequently in those classified as ALCL (1%), was detected in 23% of peripheral T-cell lymphomas not otherwise specified. Taken together, analysis of the 7q22 amplicon identified CDK6 as an important cell cycle regulator in T-cell lymphomas, representing a novel potential target for rational therapy.

MEF2C is activated by multiple mechanisms in a subset of T-acute lymphoblastic leukemia cell lines

In T-cell acute lymphoblastic leukemia (T-ALL) the cardiac homeobox gene NKX2-5 (at 5q35) is variously deregulated by regulatory elements coordinating with BCL11B (at 14q32.2), or the T-cell receptor gene TRD (at 14q11.2), respectively. NKX2-5 is normally expressed in developing spleen and heart, regulating fundamental processes, including differentiation and survival. In this study we investigated whether NKX2-5 expression in T-ALL cell lines reactivates these embryonal pathways contributing to leukemogenesis. Among 18 known targets analyzed, we identified three genes regulated by NKX2-5 in T-ALL cells, including myocyte enhancer factor 2C (MEF2C). Knockdown and overexpression assays confirmed MEF2C activation by NKX2-5 at both the RNA and protein levels. Direct interactions between NKX2-5 and GATA3 as indicated by co-immunoprecipitation data may contribute to MEF2C regulation. In T-ALL cell lines LOUCY and RPMI-8402 MEF2C expression was correlated with a 5q14 deletion, encompassing noncoding proximal gene regions. Fusion constructs with green fluorescent protein permitted subcellular detection of MEF2C protein in nuclear speckles interpretable as repression complexes. MEF2C consistently inhibits expression of NR4A1/NUR77, which regulates apoptosis via BCL2 transformation. Taken together, our data identify distinct mechanisms underlying ectopic MEF2C expression in T-ALL, either as a downstream target of NKX2-5, or via chromosomal aberrations deleting proximal gene regions.

Effects of FLT3 Ligand on Proliferation and Survival of Myeloid Leukemia Cells

FLT3 ligand (FL) acting through its tyrosine kinase receptor FLT3 has pleiotropic and potent effects on hematopoietic cells. The well-described involvement of this ligand-receptor pair in physiological hematopoiesis raised the question whether FL and FLT3 also play a role in the pathobiology of leukemia. Following the early discovery of high receptor expression by myeloid leukemia cells, several investigators have focused their attention on these cells, both primary acute myeloid leukemia (AML) cells and continuous human myeloid leukemia cell lines. Regardless of the morphological FAB subtype, the vast majority of AML cases were FLT3-positive both at the mRNA and protein level; among the myeloid cell lines, predominantly the monocytic and myelocytic cell lines were FLT3-positive whereas the erythrocytic and megakaryocytic cell lines were FLT3-negative. Virtually all cell lines studied expressed FL transcripts; the finding that some cell lines displayed both ligand and receptor indicates the possibility of autocrine, intracrine or paracrine stimulatory loops. In vitro growth assays showed that FL caused a proliferative response in a high percentage of AML cases. Only constitutively growth factor-dependent myelocytic cell lines increased their proliferation upon incubation with FL whereas all growth factor-independent cell lines were refractory to FL stimulation. Combinations of FL with various cytokines (e.g. G-CSF, GM-CSF, IL-3, M-CSF, PIXY-321, SCF) had synergistic or additive mitogenic effects. Finally, FL had significant anti-apoptotic, survival-promoting effects on primary AML cells and myeloid cell lines under serum-free culture conditions. On the strength of the above findings, it can be concluded that the FL-FLT3 signaling system may play a certain, albeit probably not causal role in the development of human leukemias. Dissection of the exact molecular pathways that lead to proliferation and/or anti-apoptosis of myeloid leukemia cells as well as the detailed elucidation of the possible contribution of the FL-FLT3 genes to leukemogenesis remain future challenges.

Elimination of mycoplasma from leukemia-lymphoma cell lines using antibiotics.

Mycoplasmal contamination of cell culture systems continues to present major problems for basic research and for manufacturing of bioproducts. Previous work suggested that certain antibiotics have strong anti-mycoplasma properties and raised the prospect that the technically rather simple antibiotic treatment may be an appropriate means for mycoplasma eradication. We have developed and validated an effective strategy to eliminate mycoplasma from chronically infected cell cultures using antibiotics which have shown strong activity against these contaminants. Here, we describe our experience with the treatment of 123 consecutive mycoplasma-positive leukemia–lymphoma cell lines, comparing five different antibiotic regimens (in total 433 treatments). We optimized the antibiotic dose schedules and the duration of treatments. The various antibiotic treatments which were employed in parallel had a high efficacy, as 71% to 86% of the infected cultures were cleansed. Treatment failure may result from the resistance of the mycoplasmas to antibiotic therapy and the inability of the eukaryotic cells to survive the cytotoxic effects of the antibiotics. Resistance to mycoplasma eradication was observed in 3% to 20% of the cultures. Loss of the cell culture caused by cytotoxicity was seen in 3% to 11% of the treatments. With regard to the overall outcome, 96% of the cell lines were rendered mycoplasma-free with at least one of the antibiotic treatments and were permanently cured. In conclusion, antibiotic treatment represents the most practical and efficient option to cleanse mycoplasma-positive cell lines.

Expression of receptor tyrosine kinase HTK (hepatoma transmembrane kinase) and HTK ligand by human leukemia-lymphoma cell lines.

HTK (hepatoma transmembrane kinase) is a receptor tyrosine kinase belonging to the EPH subfamily of tyrosine kinases. Binding of its ligand (HTKL) results in tyrosine phosphorylation of HTK. In the present study, we analyzed the possible involvement of this ligand-receptor signalling system in hematopoiesis by examining the expression of both HTK and HTKL in a large and comprehensive panel of 70 continuous human leukemia-lymphoma cell lines. HTK and HTKL mRNA expression were analyzed by reverse transcriptase-polymerase chain reaction (RT-PCR). HTK mRNA was detected in 68/70 cell lines; 58/70 cell lines were positive for HTKL mRNA expression; consequently, co-expression of both receptor and ligand was demonstrated in the majority of cell lines. Collectively, the wide-spread expression suggests a role for this ligand-receptor pair in hematopoietic development and/or function. Investigation of the details of signal transduction pathway that is activated by the HTK tyrosine kinase will help to define the exact biological function of the HTK-HTKL system.

Constitutive protein expression of monocyte chemotactic protein-1 (MCP-1) by myelomonocytic cell lines and regulation of the secretion by anti- and proinflammatory stimuli

We have investigated the protein expression of the chemokine monocyte chemotactic/chemoattractant protein-1 (MCP-1) in various human myelomonocytic leukemia cell lines. Applying specific ELISA, we demonstrated that this chemokine is produced constitutively by the cell lines HL-60, ML-2, MONO-MAC-6 and MUTZ-3 ranging between 440 and 1400 pg/ml MCP-1 per million cells. In the culture medium of two other unstimulated cell lines, MONO-MAC-1 and THP-1, almost no MCP-1 was detected. Stimulation of HL-60 and MONO-MAC-6 with lipopolysaccharide (LPS), and stimulation of ML-2 and MUTZ-3 with 12-tetradecanoyl phorbol 13-acetate (TPA) dramatically increased the MCP-1 level in the culture medium. The highest amount of MCP-1 (> 80 ng/ml within 24 h) was achieved by TPA stimulation of MUTZ-3 cells. Out of 15 cytokines tested for induction or enhancement of MCP-1 secretion, interleukin-3 (IL-3), IL-6, interferon-gamma (IFN-gamma), granulocyte-macrophage colony-stimulating factor (GM-CSF), macrophage colony-stimulating factor (M-CSF) and tumor necrosis factor (TNFalpha) were able to augment (twofold to 12-fold) the MCP-1 level in the culture medium of MONO-MAC-6 cells. While the antinflammatory cytokines IL-4, IL-10 and IL-13 failed to suppress MCP-1 secretion, the glucocorticoid dexamethasone strongly inhibited the MCP-1 production of unstimulated and stimulated MONO-MAC-6 cells. Thus, several regulatory elements are involved in MCP-1 secretion. Despite the quantitative differences of MCP-1 production among the cell lines analyzed, our results demonstrated a constitutive secretion in differentiation-arrested myelomonocytic leukemia cell lines and emphasize the usefulness of these malignant cell lines as models to study MCP-1 secretion and regulation.

1H-cyclopenta[b]benzofuran lignans from Aglaia species inhibit cell proliferation and alter cell cycle distribution in human monocytic leukemia cell lines.

Thirteen naturally occurring 1H-cyclopenta[b]benzofuran lignans of the rocaglamide type as well as one naturally occurring aglain congener all of them isolated from three Aglaia species (Aglaia duperreana, A. oligophylla and A. spectabilis) collected in Vietnam were studied for their antiproliferative effects using the human monocytic leukemia cell lines MONO-MAC-1 and MONO-MAC-6. Only rocaglamide type compounds showed significant inhibition of [3H-]thymidine incorporation and the most active compound didesmethylrocaglamide inhibited cell growth in a similar concentration range as the well-known anticancer drug vinblastine sulfate. Detailed structure-activity analysis indicated that the OH-group at C-8b which is a common structural feature of most naturally occurring rocaglamide compounds is essential for the described antiproliferative activity since replacement of this group by methylation led to a complete loss of the inhibitory activity for the resulting derivative. Rocaglamide derivatives rapidly inhibited DNA as well as protein biosynthesis of MONOMAC- 6 cells at concentrations well below those of actinomycin D or cycloheximide which were used as positive controls in the respective experiments. Didesmethylrocaglamide was furthermore able to induce growth arrest of MONO-MAC-1 cells in the G2/M and probably G0/Gl-phase of the cell cycle with no morphological indication of cellular damage. Our data suggests that 1H-cyclopenta[b]benzofuran lignans of the rocaglamide type act primarily by a cytostatic mechanism.

FLT3 Ligand Inhibits Apoptosis and Promotes Survival of Myeloid Leukemia Cell Lines

Growth factors (cytokines) are considered to be key regulators of hematopoiesis, in particular by stimulating growth or maintaining viability mainly of progenitor cells, but also of more mature cells. We examined cytokine-stimulated survival of constitutively growth factor-dependent acute myeloid leukemia (AML)-derived cell lines. The cells from the four cell lines MUTZ-2 (AML M2-derived), OCI/AML5 (AML M4), TF-1 (AML M6) and UT-7 (AML M7) undergo apoptosis quickly in the absence of cytokines in serum-free medium: half-lives of serum- and factor-deprived cells ranged from 14 to 64 h. Here, we analyzed the survival-promoting and apoptosis-inhibiting properties of FLT3 ligand (FL) using the viable cell count as an indicator of programmed cell death. The receptor for FL belongs to the class III family of receptor tyrosine kinases which also includes c-kit, the receptor for stem cell factor (SCF). FL extended the survival of cell lines MUTZ-2 and OCI/AML5, but was not effective for cell lines TF-1 and UT-7. In OCI/AML5, the action of FL was evident both in first promoting survival and then stimulating proliferation slightly. In MUTZ-2, depending on the concentration used, FL extended survival by 64-135% compared with control cells. SCF alone prolonged cell survival of MUTZ-2 as well, however, FL and the combination of FL+SCF was significantly more active. Thus, FL alone, and in combination with SCF, was active in promoting survival and proliferation of human AML cells in vitro.

Polycomb repressor complex 2 regulates HOXA9 and HOXA10, activating ID2 in NK/T-cell lines

BackgroundNK- and T-cells are closely related lymphocytes, originating from the same early progenitor cells during hematopoiesis. In these differentiation processes deregulation of developmental genes may contribute to leukemogenesis. Here, we compared expression profiles of NK- and T-cell lines for identification of aberrantly expressed genes in T-cell acute lymphoblastic leukemia (T-ALL) which physiologically regulate the differentiation program of the NK-cell lineage.ResultsThis analysis showed high expression levels of HOXA9, HOXA10 and ID2 in NK-cell lines in addition to T-cell line LOUCY, suggesting leukemic deregulation therein. Overexpression experiments, chromatin immuno-precipitation and promoter analysis demonstrated that HOXA9 and HOXA10 directly activated expression of ID2. Concomitantly elevated expression levels of HOXA9 and HOXA10 together with ID2 in cell lines containing MLL translocations confirmed this form of regulation in both ALL and acute myeloid leukemia. Overexpression of HOXA9, HOXA10 or ID2 resulted in repressed expression of apoptosis factor BIM. Furthermore, profiling data of genes coding for chromatin regulators of homeobox genes, including components of polycomb repressor complex 2 (PRC2), indicated lacking expression of EZH2 in LOUCY and exclusive expression of HOP in NK-cell lines. Subsequent treatment of T-cell lines JURKAT and LOUCY with DZNep, an inhibitor of EZH2/PRC2, resulted in elevated and unchanged HOXA9/10 expression levels, respectively. Moreover, siRNA-mediated knockdown of EZH2 in JURKAT enhanced HOXA10 expression, confirming HOXA10-repression by EZH2. Additionally, profiling data and overexpression analysis indicated that reduced expression of E2F cofactor TFDP1 contributed to the lack of EZH2 in LOUCY. Forced expression of HOP in JURKAT cells resulted in reduced HOXA10 and ID2 expression levels, suggesting enhancement of PRC2 repression.ConclusionsOur results show that major differentiation factors of the NK-cell lineage, including HOXA9, HOXA10 and ID2, were (de)regulated via PRC2 which therefore contributes to T-cell leukemogenesis.