Down-Regulation of Interleukin-8 Gene Expression in HL60 Cell Line by Human Kunitz-Type Trypsin Inhibitor

Abstract Abstract 3503 Background CD95/Fas, a member of the death receptor family, can induce cell death upon binding of its ligand, FasL Suppression of Fas signaling leads to autoimmune diseases while overexpression of Fas and FasL accounts for inappropriate apoptosis of erythroid cells in low grade myelodysplastic syndromes (MDS). The progression of MDS into secondary acute myeloid leukaemia (sAML) is associated with inhibition of hematopoietic progenitor cell death which involves the methylation of FAS gene promoter. We have previously demonstrated that FAS gene expression was increased in MDS and down-regulated when disease progresses to sAML. We further investigated the molecular mechanisms of FAS gene extinction during MDS progression. The transcription factor NFκB is functionnal as homo or heterodimer and is composed of proteins p65 or p50. The constitutive activation of NFkB pathway identified in solid tumors and in diverse hematological malignancies was suggested to promote tumor cell survival. The aim of the present work was to further investigate the interplay between FAS gene and NFkB during the progression of MDS to sAML. Methods 85 consecutive patients with a diagnosis of MDS and 15 age-matched controls with normal bone marrow (BM) were included between 2005 and 2011. Using cell lines and primary normal and leukemic bone marrow cells, we explored the p65/RelA NFkB subunit localisation by immunofluorescence and immunoblotting, inhibited NFkB pathway with the IKKa inhibitor BAY11–7082, quantified FAS transcript and protein by RT-qPCR and flow cytometry respectively, and explored p65/RelA binding to DNA by pull-down and chromatin immunoprecipitation (ChIP). Results In order to explore the role of NFkB in FAS gene expression, we used the HL-60 human leukemic cells which spontaneously express Fas at their surface and the SW-480 colon carcinoma cells which do not. We show that TNFα increased FAS gene and Fas receptor expression only in HL-60 cells indicating a cell type-dependent regulation of FAS gene expression. We conclude that NFkB is responsible for FAS gene expression in HL60 cell line. The FAS gene promoter contains putative NFκB sites in the 5' UTR region spreading between −899 and the transcription start site. By using in vitro DNA affinity precipitation experiments, we show that P65/RelA bound to each of the 3 putative NFκB binding sites on FAS promoter upon TNFa treatment. We conclude that p65/RelA binds to the chromatin at FAS promoter in a cell type-dependent manner to regulate FAS gene expression in cells. Then, we hypothesized that DNA methylation in FAS gene 5'UTR regulatory regions may prevent the docking of NFkB. ChIP Experiments identified one active mark H3K9/14Ac and none of the two repressive marks H3K27me3 and H3K9me2 in HL60 cell line while the repressive mark H3K9me2 was expressed in SW480 cell line These results suggested that the chromatin conformation at FAS promoter was open in Fas-positive HL-60 cells, and repressed in Fas-negative SW-480 cells. By treatment with azacitidine, which stimulated the expression of Fas in SW-480 but not in HL-60 cells and by experiments using TNFa, we show that NFkB-dependent FAS gene expression is epigenetically regulated. We used ChIP to explore the binding of p65/RelA to the chromatin at FAS promoter in 4 Fas-expressing and 6 Fas-negative MDS samples. The expression of Fas was proportional to the recruitment of p65/RelA at the gene promoter. Among 16 MDS samples where FAS protein expression was positive, BAY11–7082 was able to decrease Fas expression in 12 of them. In addition, we show that azacitidine rescues NFκB-induced Fas expression in primary MDS/sAML cells. Altogether, NFκB controls the transcription of FAS gene in primary BM cells from MDS or sAML and azacitidine can induce FAS promoter demethylation, thus facilitates p65/RelA binding and FAS gene transcription in myeloid cells. Conclusion The present study demonstrates that NF-kB directly promotes FAS gene expression in normal and myelodysplastic hematopoietic progenitor cells and that, when MDS progress into sAML. The inhibition of FAS gene expression observed in sAML is due to the methylation of NFkB binding site in the gene promoter, which can be reversed by the demethylating agent 5-azacitidine. The increased expression of FAS gene induced by 5-azacitidine now deserves to be tested as a biomarker of the drug activity. Disclosures: No relevant conflicts of interest to declare.

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Effect of Second Mitochondria-Derived Activator of Caspases (Smac) Mimetic Compounds on Leukemic Cell Lines.

Blood ◽

10.1182/blood.v112.11.1609.1609 ◽

2008 ◽

Vol 112 (11) ◽

pp. 1609-1609

Author(s):

Federica Servida ◽

Francesco Onida ◽

Domenico Delia ◽

Cinzia Scavullo ◽

Daniele Lecis ◽

...

Keyword(s):

Cell Line ◽

Cell Lines ◽

Cytotoxic Effect ◽

Leukemic Cell ◽

Hl60 Cell ◽

Caspase 9 ◽

Leukemic Cell Lines ◽

Smac Mimetics ◽

Smac Mimetic ◽

Hl60 Cell Line

Abstract The apoptotic process and its dysfunctions have become the focus of extensive pharmaceutical research in solid and hematopoietic tumors as well as neurodegenerative diseases. The X-Inhibitor of Apoptosis Protein (XIAP) binds caspase 9, 3 and 7, preventing their activation and, consequently, apoptosis. The Smac/DIABLO protein, released from mitochondria, binds XIAP as a dimer on the same caspase 9 (BIR3 domain) binding site. Similarly, the Smac protein interferes with the XIAP binding site for caspases 3 and 7, thus promoting both the extrinsic and intrinsic apoptotic paths. The thin balance of this binding equilibrium is altered in various tumors, including leukemia, where XIAP is overexpressed and a caspase-dependent resistance to enter apoptosis is usually observed. Thus, XIAP inhibition via Smac mimetics’ binding is at the same time a characterized protein-protein interaction, and a validated mechanism for intervention in cancer therapy. We tested 56 Smac mimetic compounds (designed by CISI - Center for biomolecular Interdisciplinary Studies and Industrial applications of the Milan University) for their in vitro capacity to bind to the XIAP BIR3 domain. We also evaluated the ability of the Smac mimetic compounds to inhibit the growth of the human leukemia HL60, K562 and Jurkat cell lines (derived from patients with promyelocyitic leukemia, blastic phase-CML and T acute lymphoblastic leukemia, respectively). Nine compounds which were shown to be active, were further investigated for their effect on cell cycle (by DNA staining with propidium iodide and cytofluorimetric analysis) and for possible synergistic effect in combination with other chemotherapeutic drugs (Cytarabine, Etoposide and Idarubicine). The same compounds were also tested on normal CD34+ hematopoietic progenitor cells. The cytotoxicity was evaluated after 72 hours treatment with Smac mimetic compounds by a colorimetric assay for the quantification of cell proliferation and viability based on the cleavage of the WST-8 tetrazolium salt by mitochondrial dehydrogenases. The effect of Smac mimetic compounds on CD34+ cells enriched from mobilized peripheral blood was assessed as the capability of inhibiting the myeloid colony growth (CFU-GM). The data were expressed as mean percentage of 3 replicates normalized to the untreated control. Overall, a strong correlation between the binding affinity to the XIAP BIR3 domain and the cytotoxic effect on the leukemic cell lines was observed. The more promising compounds showed IC50 ranging from 0,3 to 1 microM on the HL60 cell line. The Jurkat and K562 cell lines were less sensitive, with IC50 ranging from 11,8 microM to more than 50 microM. However, in the K562 cell line, the combined treatment unveiled synergistic effect with Cytarabine and Etoposide (R Kern index = 1,4 and 1,5 respectively). No cytotoxic effect was observed on normal controls at doses up to 80 microM. A consistent sub G1 apoptotic peak (up to 53% of apoptotic cells) was observed in the HL60 cell line after 48 hrs treatment, thus suggesting a strong activation of the apoptotic process. All together, our data suggest that Smac mimetics may have a promising therapeutic potential as a new class of anticancer drugs in hematopoietic malignancies. Further experiments are currently ongoing to confirm the effectiveness of these compounds also on primary cells from leukemia patients, both as single agents and in combination with conventional drugs. In particular, due to their ability to enhance pro-apoptotic effect, Smac mimetic compounds may allow to overcome resistance of cancer cells to standard chemotherapy.

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