Cell Lines KG-1 Research Articles

Abrogation of the DNA-Damage Response in De Novo AML Versus MDS Cell Lines.

Background: The increasing evidence that activation of the DNA-damage-response (DDR) pathway in preneoplastic lesions of solid neoplasms hinders tumorigenesis prompted us to evaluate the same hypothesis in cell line models representing different subtypes of MDS or AML.Methods: myeloid cell lines corresponding to different subgroups of MDS, having or not progressed to AML (p39, MOLM-13) and de novo AML (MV4-11, and KG-1) were assessed for their capacity to induce a DDR. Using the quintessential inducer of a DDR, the cell lines were gamma-irradiated (2, 5 and 10Gy) and their ability to arrest the cell cycle in G2/M and to undergo apoptosis were assessed.Results: in contrast to the de novo AML-derived KG-1 cells - the irradiated MDS/AML-derived cell lines P39 and MOLM-13 exhibited an earlier and more pronounced arrest of the cell cycle in G2/M, as well as a higher percentage of apoptotic cells. Whereas 10 Gy induced a G2/M-arrest in about 30% of KG-1 cells, this percentage increased to about 60% in P39 and MOLM-13 cells. Comparably, whereas KG-1 cells exhibited about 15% of apoptotic cells following irradiation with 10Gy, the percentage reached 30% in P39 and MOLM-13 cells. Assessing the underlying molecular determinants we demonstrated that irradiated P39 and MOLM-13 cells retained the ability to activate crucial mediators of the DDR, whereas this ability was decreased in the de novo AML cell line KG-1. In particular, in comparison to KG-1 cells, irradiated P39 and MOLM-13 cells exhibited a more pronounced upregulation of the crucial cell cycle/checkpoint regulators Chk1-P-Ser317 and Cdk1-P-Tyr15. Noteworthy, MDS cell lines with an increased G2/M-arrest exhibited a higher expression of nucleophosmin - which serves as a shuttling protein between the nucleus and the cytoplasm - and were able to retain this increased expression upon irradiation. Moreover, elevated expression of NPM in MDS cell lines corresponded to higher PCNA-expression, which serves as an essential component of the DNA-repair machinery. To further investigate the functionality of the DDR-pathway, the different cell lines were gamma-irradiated in the presence of the ATM-inhibitor KU-55933 and the Chk1 inhibitor UCN-01. Whereas inhibition of Chk1 (and to a lesser extent inhibition of ATM) was able to hinder G2/M-progression in irradiated MDS cells, it remained without effect in the AML cell lines KG-1 and MV4-11. Noteworthy, whereas irradiated P39/MDS cells hindered to activate Chk1 decrease expression of Polo-like-kinase-1 (Plk-1) - which not only governs mitotic entry and exit, but also functions as an oncogene - KG-1 cells maintained a high expression of Plk-1, comparable to that observed in non-irradiated cells.Conclusion: we demonstrated the different capacities of MDS (or AML post MDS) and de novo AML cell lines to activate the DDR and thus provide evidence that the DDR-pathway is abrogated in de novo AML as compared to MDS cells.

Reversal of p15/ INK4b hypermethylation in AML1/ETO-positive and -negative myeloid leukemia cell lines

In vitro and in vivo, myeloid leukemic and preleukemic cells exhibit variable sensitivity to the antiproliferative and proapoptotic effects induced already at low concentrations of DNA methyltransferase (DNMT) inhibitors. The molecular mechanisms underlying this variable sensitivity of leukemic blasts to azanucleosides such as 5-azacytidine and 5-aza-2′-deoxycytidine (DAC) may involve modifier effects of specific fusion proteins such as AML1/ETO. The cyclin-dependent kinase inhibitor p15/ INK4b is one potential target of DNA demethylating activity in AML and MDS where it is frequently silenced by hypermethylation. To study sensitivity to DAC in myeloid leukemia cells, we chose the myeloid cell lines Kasumi-1 (expressing AML1/ETO), KG-1 and KG-1a (both AML1/ETO-negative) all of which a highly methylated p15/ INK4b gene. Treatment with DAC resulted in dose-dependent regional demethylation of p15/ INK4b in Kasumi-1 and KG-1, but only to a modest degree in KG-1a cells. Demethylation was associated with induction of p15/INK4b protein expression. Growth-inhibitory and proapoptotic activity of DAC was significantly higher in Kasumi-1 than in KG-1a cells, and sensitization of cells to a cooperating effect of All-trans retinoic acid and of the histone deacetylase (HDAC) inhibitor Trichostatin A was observed. DAC-induced growth inhibition and apoptosis were enhanced when AML1/ETO was conditionally expressed in AML1/ETO-negative U-937 cells. In conclusion, hypomethylation and reactivation of p15/ INK4b in myeloid cell lines are among the molecular events associated with DAC-induced growth arrest and apoptosis. Further studies of AML1/ETO as a modifier of the epigenotype and sensitivity of myeloid cells to inhibitors of DNMTs and HDACs appear warranted.

Immunosuppressive activity of recombinant ILT3

Tolerogenic antigen presenting cells (APC) are characterized by high expression of the inhibitory receptors ILT3 and ILT4. We have engineered ILT3 and ILT4 cytoplasmic deletion mutants (ILT3delta and ILT4delta), which were transfected in the dendritic-like cell line KG1, to investigate ILT3 and ILT4's capacity to signal extracellularly. KG1.ILT3delta, similar to untruncated ILT3, inhibits T cell responses such as proliferation and cell-mediated cytotoxicity. In contrast, KG1.ILT4delta lost the suppressive activity of untruncated ILT4. This indicates that the inhibitory function of ILT4 relies entirely on the cytoplasmic region containing ITIM motifs. We further demonstrated that recombinant soluble ILT3 inhibits T helper and cytotoxic function while inducing the differentiation of CD8 + Ts cells. Hence, Ts modulate APC function inducing inhibitory receptors, which in turn elicit the generation of Ts.

Marked synergism of cytotoxic agents with either a prenylation inhibitor or M-TOR inhibitor in acute leukemia cell lines: Potential clinical implications

13103 Background: The most combinations of anticancer drugs are based upon empiricism. The potential permutations of drugs overwhelm the clinical trials system. Acute leukemia is sensitive to a variety of agents but relapses are common. Targeted agents are attractive new venues of therapy both as single agents and in combination with older agents. Isobologram median effect analysis allows up to three agents to be studied together in vitro to identify interesting combinations. We evaluated a commercially available statin, fluvastatin, to block prenylation which affects a variety of pathways, rapamycin and its experimental analogue RAD001 as M-TOR inhibitors to block downstream of the AKT pathway, and cytotoxic agents. Methods: The human leukemia cell lines AML-193 and KG-1 were obtained from ATTC (Rockville, MD), fluvastatin and RAD001 from Novartis Pharma, and the other agents from Sigma-Aldrich (St. Louis, MO). The IC50 of the single agent was determined by a 72 hr incubation of log growth cells using a MTT assay and the EZ-ED50 program (Perrella Scientific, Conyers, CA). The dosages of all agents were at clinically achievable concentrations. All reported values were the means of at least 3 experiments with each study using 4 wells per point. For isobologram analysis, a minimum of 8 concentrations of drug mixtures were studied above and below the IC50. Median effect CI values less than 1 are synergistic. Results: Doublets of fluvastatin with Ara-C (0.7), daunomycin (0.4), idarubicin (0.7), RAD001 (0.5), or rapamycin (0.3) demonstrated synergy. Doublets of RAD001 with Ara-C (0.3), daunomycin (0.7), or idarubicin (0.5) demonstrated synergy. Triplets of RAD001/daunorubicin/Ara-C, RAD001/daunomycin/fluvastatin, and RAD001/Ara-C/idarubicin all demonstrated marked synergy in both cell lines. Conclusion: A new potential non classical combination for further investigation is RAD001 or rapamycin with an inhibitor of prenylation such as fluvastatin. Additional potential combinations include cytotoxics with either fluvastatin or RAD001, and triplet combinations. No significant financial relationships to disclose.

Production and secretion of human interleukin-18 in transgenic tobacco cell suspension culture

Interleukin-18 (IL-18), otherwise known as interferon-gamma-inducing factor (IGIF), is one of several well characterized and important cytokines that contribute to host defenses. The complementary DNA (cDNA) of mature human interleukin-18 gene (hIL-18) was fused with the signal peptide of the rice amylase 1A gene (Ramy1A) and introduced into the plant expression vector under the control of a duplicated CaMV 355 promoter. The recombinant plasmid was transformed into tobacco (Nicotiana tabacum L. cv Havana) using theAgrobacterium-mediated transformation method. The integration of the hIL-18 gene into the genome of transgenic tobacco plants was confirmed by polymerase chain reaction (PCR) amplification and its expression was observed in the suspension cells that were derived from the transgenic plant callus by using Northern blot analysis. The hIL-18 protein was detected in the extracts of the transgenic callus and in the medium of the transgenic tobacco suspension culture by using immunoblot analysis. Based upon enzyme-linked immunosorbant assay (ELISA) results, the expression level of the hIL-18 protein approximated 166 μg/L in the suspension culture medium. Bioassay results from the induction of interferon-γ from a KG-1 cell line indicated that the hIL-18 secreted into the suspension culture medium was bioactive.

Switching Death Pathways in AML; Induction of Necrosis by Arsenic, Ascorbic Acid and Antabuse (AAA).

Virtually all chemotherapy drugs induce apoptosis. Cancer cells have multiple mechanisms of resistance to this form of cell death. Necrosis may have fewer mechanisms of resistance, providing a rationale to discover ways to switch cell death pathways from apoptosis to necrosis. Arsenic trioxide (ATO) is clinically effective against acute promyelocytic leukemia and studies have suggested efficacy in treating other malignancies, including other leukemias. ATO increases the intracellular concentration of reactive oxygen species and inhibits glutathione (GSH) reduction resulting in redox changes which activate caspases and induce apoptosis. Ascorbic acid (AA) and other agents which reduce glutathione concentrations and increase hydrogen peroxide concentrations have been shown to enhance the antitumor activity of ATO in vitro. This combination of agents is currently in clinical trials. In previous experiments using pancreatic cancer cell lines, disulfiram (DSF), in clinical use as an inhibitor of aldehyde dehydrogenasel, was added to the ATO/AA combination. Synergistic induction of cell death occurred. We sought to test this combination in myeloid cell lines. We tested the ATO/AA/DSF (AAA) combination ([ATO], 1uM; [AA], 100uM; [DSF], 0.125 uM) on two myeloid leukemia cell lines, KG1 and K562. After treatment for 48 hours in AAA, annexin and propidium iodide staining revealed an increase in annexin(+) PI(+) cells (necrosis) in the KG1 cell line that was similar to the effect in pancreatic cancer cells. Nearly five fold more KG1 cells underwent necrosis after exposure to AAA than with arsenic alone. Arsenic combined with ascorbic acid or disulfiram yielded an approximately two fold increase in necrosis. We propose that the mechanism of action of disulfiram is through quenching peroxide (H2O2) radicals generated by ATO and AA. The reducion of DSF leads to oxidation of GSH and NADH which decrease [ATP]i, shifting the cells from apoptosis to necrosis. K562 cells were largely resistant to the triple combination, remaining in a state of cytostasis after exposure to AAA. BCR-ABL positive cell lines, such as K562, have a relative increase in intracellular ROS. The lack of necrosis in K562 was surprising, and suggests that the activated BCR-ABL kinase may override this proposed mechanism. Imatinib has been shown to induce necrosis, and combinations including imatinib will be tested in BCR-ABL positive cell lines. To study the potential safety of this combination for human trials, AAA was tested on peripheral blood stem cells from normal donors (obtained after informed consent) and did not appear to have synergistic effects on colony formation as compared to the drugs applied individually. These results suggest a new mechanism of cell death, switching from apoptosis to necrosis, that can be targeted to treat leukemias and other malignancies. More studies to confirm the mechanism of this cell death are underway.

Nucleoside Analogues of Cladribine Produce Enhanced Responses in Cell Lines.

Nucleoside analogues are antimetabolites that can be used to treat AML. These compounds mimic the effects of physiological nucleotides in terms of uptake and metabolism and become incorporated into nascent DNA resulting in synthesis inhibition and chain termination. Nucleoside drugs often suffer from poor metabolism to the active triphosphate forms, deactivation and clinical toxicities. Phosphate prodrugs (protides) have modified monophosphate groups on the drug which potentially enables them to enter cells and subsequently become metabolised into the monophosphate form. Production of the monophosphate form of the drug is the rate limiting step in the production of the active triphosphate form.Cladribine (2-chlorodeoxyadenosine) has been used as a parent drug to produce a series of protides with altered monophosphate groups. The parent drug and the protides were used at concentrations between 10μM to 0.002μM on a panel of six myeloid cell lines (HL-60, NB4, NB4R2, U937, KG1 and K562) for 72 hours. The effects of the protides were measured by proliferative assays (MTS) and IC50 values calculated. Two protides, CC122 and CC124, were assessed in combination with cytarabine (Ara-C) at ratios of 1:1 and 1:10 in the same manner as previously described.Fourteen protides of cladribine were analysed in addition to cladribine itself and Ara-C. The average IC50 value for each protide, across all the cell lines, tested were reduced by 2 to 11 fold compared to cladribine and Ara-C. One protide (CPF193) had no effect on any of the cell lines, whilst CPF194 was found only to be active in the KG1 cell line with a 2.7 fold reduction in the concentration required kill 50% of the cells treated compared to cladribine. In general, K562 cells did not respond to treatment with Ara-C, cladribine or 6 of the protides although they did respond to the remaining protides with IC50 values slightly higher than the other cell lines.The IC50 values for each protide in each cell line individually was also calculated and some cell lines, particularly NB4, showed dramatic reductions in IC50. In particular protide CC124 showed over 40-fold reduction from 9.4μM for cladribine to 0.23μM for CC124. This was also lower than the IC50 for Ara-C in this cell line (0.53μM).Ara-C and cladribine combinations, at a ratio of 1:1, gave an average combination index (CI) at the IC50 of 0.9857 across all the cell lines, excluding K562, indicating an additive effect only. However, protide CC124 and Ara-C in combination, at the same ratio, produced an average CI of 0.6334 indicating synergistic activity.This study has shown that altered monophosphate groups on the nucleoside inhibitor cladribine reduces the IC50, particularly when used in combination with Ara-C. The reduced doses would be beneficial in a clinical environment.

Raf Inhibitor BAY 43-9006 Induces Bim Dephosphorylation and Activates the Intracellular Apoptotic Pathway in AML.

We have previously demonstrated constitutive activation of MAPK signaling in 70% of primary AML samples (Millela et al, JCI 108:851–859, 2001), suggesting that upstream kinases (Raf and MEK) may play a role in the leukemic transformation of myeloid cells. BAY 43-9006 is a small molecule Raf kinase inhibitor that has demonstrated potent anti-tumor activity against solid human tumors in xenograft models. In this study, we tested the hypothesis that BAY 43-9006 inhibits leukemia cell growth and/or induces apoptosis by suppressing the activity of the MAPK pathway. In the in vitro kinase assay, BAY 43-9006 inhibited both Raf-1 and B-Raf-mediated MEK1 phosphorylation in a dose-dependent manner, with Raf-1 kinase being more sensitive to the inhibitory effects of BAY 43-9006 (IC50Raf-1, 1.37 μM vs. IC50B-Raf, 4.64 μM). BAY 43-9006 suppressed MEK1/2 and ERK phosphorylation in the AML cell lines OCI-AML3, HL-60, U937 and KG-1 in a dose-dependent manner after 24 hr treatment. Unexpectedly, BAY 43-9006 also inhibited AKT phosphorylation on Ser473 (after 4.5 hrs). BAY 43-9006 inhibited growth of AML cells in a dose- and time-dependent manner. The 50% inhibitory concentration (IC50) of BAY 43-9006 was 0.39, 1.14, 2.86 and 2.80 μM, respectively in OCI-AML3, HL-60, U937 and KG-1 cells after 72 hrs. This growth-inhibitory effect was mediated by a dose-dependent induction of cell cycle arrest in G1 mediated by the down-regulation of the cell cycle-related proteins cyclin E, cdk2 and cdc2, followed by induction of apoptosis after 72 hrs. In primary AML patient samples, BAY 43-9006 not only inhibited cell growth and induced apoptosis after 48–72 hrs in vitro, but also preferentially inhibited colony formation of AML progenitor cells compared to normal bone marrow cells [IC50: 2.33 μM vs. 9.34μM (CFU-GM), 5.69 μM (Erythroid) and 3.75 μM (Mixed), respectively]. Time-course analyses demonstrated that BAY 43-9006 suppressed phosphorylation of the pro-apoptotic protein Bim (at 4.5 hrs), caused loss of the mitochondrial membrane potential and cytochrome c release (at 6 hrs) followed by cleavage of caspases-3 and -9 but not of caspase-8, suggesting primary involvement of the intrinsic mitochondrial pathway. Furthermore, the pro-apoptotic proteins Bim and Bax were up-regulated after 48 hrs of BAY 43-9006 treatment, and the level of the inhibitor-of-apoptosis protein Survivin was down-regulated after 48 hrs. In summary, our data demonstrates that BAY 43-9006 inhibits Raf-MEK-ERK signaling and induces apoptosis in AML via Bim de-phosphorylation and activation of the intrinsic apoptotic pathway. The potential of BAY 43-9006 in the therapy of AML patients will be tested in a Phase I clinical trial.

Novel Synthetic Histone Deacetylase Inhibitor (SK-7041) Potently Inhibits Proliferation in Acute Myeloid Leukemia Cell Lines.

Histone acetyltransferase (HAT) and histone deacetylase (HDAC) activities determine the acetylation status of histones, and have the ability to regulate gene expression through chromatin remodeling. Aberrant histone acetylation is known to play a key role in leukemogenesis. To improve the treatment outcome in AML patients, we examined the antiproliferative activity of SK-7041, a novel hybrid synthetic HDACI, in acute myelogenous leukemia (AML) cell lines (KG-1,HL-60 and HEL). This HDACI preferentially inhibited the enzymatic activities of HDAC1 and HDAC2, compared to other HDAC isotypes, indicating that class I HDAC is the major target of SK-7041. SK-7041 effectively induced the time-dependent hyperacetylation of histones, H3 and H4 in AML cell lines. We found that this compound exhibited potent antiproliferative activity against AML cell lines in vitro. The growth inhibitory effect of SK-7041 was related to the induction of apoptosis in AML cell lines. SK-7041(4uM) induced potent cell death, triggering apoptosis in over 60% of cells in 48h by FACS analysis. After 12h of treatment by western analysis, apoptotic cells were increased through activation of caspase-7,-9,-3 and cleaved-PARP (caspase-8 was not affected). Cyclin D1 expression was decreased, and CDK4 expression level was not affected by SK-7041. The level of p21 expression was gradually increased in KG-1 and HL-60 cell lines, whereas treated HEL cell lines showed no change. For further investigation of the genes affected by SK-7041, 20Kb oligonucleotide microarray analysis was performed. The expression of candidate genes, which we selected in chip analysis, was confirmed using real-time PCR. Fifteen genes (RGL1, FYN, CARD9, ABCA7, TNFRSF6B, CASP9, ENPP2, etc.) were consistently up-regulated and twelve genes (PTPN7, CD34, INSIG1, IL16, LHX6, TRIB3, BID, PDCD4, etc.) were down-regulated more than 4-fold by the SK-7041 compound. We suggest that the efficacy of SK-7041 was mainly mediated by the inhibition of proliferation and induction of apoptosis. Taken together, our data suggested that novel HDACI SK-7041 treatment is effective in AML cell lines, and that the further study is warranted.

High FLT3 Transcript Level Is Associated with Unfavorable Prognosis in Pediatric Non-Promyelocytic Acute Myeloid Leukemia.

FLT3 is a receptor tyrosine kinase involved in the survival of hematopoietic stem cells, and mutations of FLT3 have been reported to be a risk factor in acute myeloid leukemia (AML), especially for pediatric patients. Recently, FLT3 expression level is proposed to have a prognostic significance in adults, but little is known for childhood AML.FLT3 transcript levels at diagnosis were measured with real time quantitative reverse transcription polymerase chain reaction to verify the clinical significance in pediatric AML. FLT3 transcript levels were analyzed in 20 (M: 12, F: 8) pediatric patients with median 6 (0 – 16) years old received same treatment scheme (Leuk Res. 29; 617). Patients with secondary AML or RAEB-t were included, but patients with FAB M3 type were excluded. Relative gene expression levels were calculated by using standard curves made with KG-1 cell line copy and adjusted on the basis of the expression level of the GAPDH gene (106 copy). Each gene expression level was examined in triplicate, and the mean value was subjected to analysis. The copy number of FLT3 in each sample was calculated by comparing the Ct values of samples with standard curves. Mann-Whitney test was applied for comparison of means. For survival data, Kaplan-Meier life tables were constructed and the curves were compared by means of log-rank test.The median value of adjusted FLT copy number was 58 (0.06–19,302) x 103. FLT3 copy number was not associated with gender, age and other risk factor except FLT3 internal tandem duplication (FLT3/ITD) which was present in 3 patients with significantly higher mean copy number (7,130 x 103) than others (372 x 103) (P = 0.023). The mean value of FLT3 copy number of 12 refractory or relapsed patients (2,278 x 103) was higher than that of patients in continuous remission (48 x 103), significantly (P = 0.031). The overall survival rate of 10 patients with high FLT3 copy number more than 50 x 103 (25.0%) was lower than that of others (59.3%) significantly (P = 0.0457). All patients with high copy number were refractory to induction or relapsed early before post remission transplantation except 2 patients (one received bone marrow transplant from matched sibling and another received autologous peripheral stem cell transplant).High FLT3 transcript level was an unfavorable prognostic factor for early relapse and overall survival in pediatric AML and associated with FLT3/ITD. New therapeutic scheme such as stem cell transplantation with more intensive conditioning just after complete remission and administration of FLT3 inhibitor could be applied in pediatric non-promyelocytic AML patients with high FLT3 transcript level.

Stem Cell Mobilization and Egress of AML Blasts from the Bone Marrow Are Both Regulated by MT-MMP-Mediated Activation of MMP-2.

The cell-surface proteolytic enzymes membrane-type (MT)-matrix metalloproteinases (MMPs) activate secreted latent forms of MMPs and play a key role in cell migration and tumor cell invasion and metastasis. Previously we reported that mobilized peripheral blood (mPB) CD34+ cells as well as AML blasts secrete inactive (pro)MMP-2, in contrast to normal steady-state bone marrow (BM) CD34+ cells which do not. In this study we hypothesized that the egress from BM of normal hematopoietic stem/progenitor cells during G-CSF-induced mobilization or of AML blasts shares a common mechanism that involves activation of secreted proMMP-2 by MT-MMPs. We evaluated the expression of MT-MMPs (MT1-MMP, MT2-, MT3- MT4-, MT5-, MT6-) in normal and leukemic hematopoietic cells, as well as in BM stromal cells, and determined their role in proMMP-2 activation and migration. We found MT1-MMP expression (mRNA and protein) in mPB CD34+ cells and in the majority of AML samples (from 21 out of 26 patients) and upregulation of MT1-MMP by G-CSF in steady-state BM CD34+ cells. Moreover, we confirmed the secretion of proMMP-2 in media conditioned by hematopoietic (mPB CD34+, AML) cells and stromal cells. However, we detected the active form of MMP-2 in co-cultures of the hematopoietic (mPB CD34+, AML) cells with stromal cells, as well as in co-cultures of steady-state BM CD34+ cells stimulated with G-CSF with stroma. In cultures of stromal cells alone G-CSF had no effect on the expression of either MT1-MMP or MMP-2. To evaluate the role of MT-MMPs in proMMP activation, we examined the leukemic KG-1 cell line, which we found not to express any MT-MMPs, and did not detect active MMP-2 in co-cultures with stroma. On the other hand, primary AML samples that did not express MT1-MMP but expressed MT2-, MT4- or MT5-MMPs activated proMMP-2 in co-cultures with stroma. Moreover, the MT1-MMP inhibitor epigallocatechin-3-gallate significantly reduced trans-Matrigel migration of mPB CD34+ (by 50%) and AML cells (by 70–90%). Hence we suggest that in the BM microenvironment (i) MT1-MMP localized on the surface of hematopoietic cells activates MMP-2, inducing a highly proteolytic microenvironment, and (ii) MT1-MMP upregulation by G-CSF in BM CD34+ cells can result in CD34+ cell mobilization from BM. Similarly, the constitutive high expression of MT1-MMP and other MT-MMPs in AML blasts also contributes to MMP-2 activation in the BM microenvironment and may be conducive to the egress of AML blasts from the BM.

311 PUBLICATION Different cell cycle kinetic effects of N1N11-diethylnorspermineinduced polyamine depletion in four human breast cancer cell lines

3-Hydrogenkwadaphnin (3-HK) (Fig. 1) is a daphnane-type diterpene ester isolated from the leaves of Dendrostellera lessertii (Thymelaeaceae) with differentiation and apoptotic potency among several leukemic cells without any measurable adverse effects on normal cells [Moosavi, M.A., Yazdanparast, R., Sanati, M.H., Nejad, A.S., 2005a. 3-Hydrogenkwadaphnin targets inosine 5′-monophosphate dehydrogenase and triggers post-G1 arrest apoptosis in human leukemia cell lines. Int. J. Biochem. Cell Biol. 37, 2366–2379]. In this study, we evaluated differentiating and apoptotic efficiency of a second new anti-proliferating agent from the same plant relative to 3-HK using acute myeloid leukemia (AML) KG1 cell line. 3-HK at 5–30 nM inhibited proliferation of KG1 cells after 24–96 h of treatment. NBT reducing assay and expression of cell surface markers (CD11b and CD14) confirmed that the inhibition of proliferation is associated with differentiation toward macrophage-like morphology. Regarding the relatively weaker potency of 3-HK in the induction of differentiation compared to that of the crude extract, we looked for additional compound(s) with similar properties in the crude extract. This effort led to isolation of the second compound from the leaves’ extract with higher differentiating potency. The new compound inhibited proliferation of KG1 cells by almost 48±3.1% after 72 h of treatment with a single dose of 1.5 μg/ml. The treated cells differentiated along the monocyte/macrophage lineage based on the morphological features apparent after Wright–Giemsa staining, phagocytic activity and expression of cell surface markers as analyzed by flow cytometry. On the other hand, the results indicated that exposure of KG1 cells to either 3-HK or the new compound for 3–4 days induced apoptosis as assayed qualitatively by acridine orange/ethidium bromide (Ao/EtBr) double staining, agarose gel electrophoresis and quantitatively by Annexin-V technique and sub-G1 DNA staining using flow cytometry. Based on the present data, these two active constituents of D. lessertii have the novelty of being further evaluated for pharmaceutical applications.

Regulation of RelB Expression during the Initiation of Dendritic Cell Differentiation

The transcription factor RelB is required for proper development and function of dendritic cells (DCs), and its expression is upregulated early during differentiation from a variety of progenitors. We explored this mechanism of upregulation in the KG1 cell line model of a DC progenitor and in the differentiation-resistant KG1a subline. RelB expression is relatively higher in untreated KG1a cells but is upregulated only during differentiation of KG1 by an early enhancement of transcriptional elongation, followed by an increase in transcription initiation. Restoration of protein kinase CbetaII (PKCbetaII) expression in KG1a cells allows them to differentiate into DCs. We show that PKCbetaII also downregulated constitutive expression of NF-kappaB in KG1a-transfected cells and restores the upregulation of RelB during differentiation by increased transcriptional initiation and elongation. The two mechanisms are independent and sensitive to PKC signaling levels. Conversely, RelB upregulation was inhibited in primary human monocytes where PKCbetaII expression was knocked down by small interfering RNA targeting. Altogether, the data show that RelB expression during DC differentiation is controlled by PKCbetaII-mediated regulation of transcriptional initiation and elongation.

5-Aza-2′-deoxycytidine (decitabine) can relieve p21WAF1 repression in human acute myeloid leukemia by a mechanism involving release of histone deacetylase 1 (HDAC1) without requiring p21WAF1 promoter demethylation

Decitabine is a potent demethylating agent that exhibits clinical activity against myeloid malignancies. Numerous genes silenced by hypermethylation are reactivated by decitabine through a mechanism involving promoter demethylation with subsequent release of histone deacetylases (HDACs) and accumulation of acetylated histones. Recent studies indicating that decitabine also induces regional chromatin remodeling of some unmethylated genes suggest additional mechanisms of action. Decitabine reactivates unmethylated p21WAF1 in some AML cell lines but the possible occurrence of p21WAF1 methylation in AML in vivo has not been studied in detail and decitabine effects on p21WAF1 chromatin remodeling have not been reported. We found that p21WAF1 mRNA was undetectable in 6 of 24 AML patient samples and 4 of 5 AML cell lines but there was no evidence of p21WAF1 promoter methylation. However, decitabine induced p21WAF1 in AML cell lines KG-1 and KG-1a in association with release of HDAC1 and increased acetylated histone H3 at the unmethylated p21WAF1 promoter. Decitabine effects on p21WAF1 histone acetylation and induction were enhanced by the HDAC inhibitor trichostatin A and were independent of wild type p53. Our findings indicate that decitabine can relieve p21WAF1 repression in AML by a mechanism that involves release of HDAC1 without requiring promoter demethylation. Furthermore, our study provides evidence that combined decitabine and HDAC inhibitor treatment can enhance chromatin remodeling and reactivation of an unmethylated tumor suppressor gene. This latter finding is of relevance to the clinical use of these agents in AML as we found the p21WAF1 promoter to be unmethylated in vivo.

Monoclonal antibodies specific for the IL-18 receptor

Interleukin-18, a pleiotropic cytokine is a member of the IL-1 family and has multiple immunoregulatory functions. IL-18 action leads to IFNγ production by NK or T cells, induces Th1 differentiation and suppresses IgE synthesis by B cells when acting on responding cells in association with IL-12. At present two subunits of the IL-18R have been characterized: IL-18 Rα and IL-18 Rβ. Both receptors belong to the IL-1R family. IL-18 Rα has been described as the ligand-binding chain and IL-18 Rβ as the signal-transduction chain. Three monoclonal antibodies (mAbs) submitted to the HLDA8 workshop, designated H44 (80438), B-B46 (80228), and B-E43 (80232) were evaluated. The mAb specificity was determined by ELISA using coated recombinant IL-18 Rα or IL-18 Rβ. Cell staining was analyzed by flow cytometry. A positive staining with the mAb B-E43 or H44 demonstrated that IL-18 Rα is expressed on several myeloid cell lines. No positive cell staining was observed with the anti IL-18 Rβ mAb B-B46. The mAb biological activity was studied using the cell line KG1. A downmodulation of IFNγ production was observed with the mAbs B-B46 (80228) and B-E43 (80232).

Tumor Necrosis Factor Alpha Inhibits Telomerase and Induces Senescence in Acute Myeloid Leukemia Cells.

Tumor necrosis factor alpha (TNFα) is a multifunctional cytokine, which is overproduced in a series of hematological diseases, including acute myeloblastic leukemia (AML) and myelodysplasia (MDS). Apoptotic cell killing has been the most frequently documented cellular effect of TNFα in AML cells. Much little is known about TNFα effect in AML cells, which display an intrinsic defect in their apoptotic machinery. In this study, we describe that prolonged exposure to TNFα, but not Fas, resulted in senescence in the immature CD34+ CD38− KG1 cell line. Indeed, TNFα-treated KG1 cells displayed growth arrest, increased senescence-associated β-galactosidase activity, telomere shortening and additional chromosomal abberations. In these cells, as well as in fresh AML cells, treatment with TNFα resulted in decreased hTERT gene trancription and hTERT activity. Moreover, we found that ceramide production and JNK activation were critical signaling components in TNFα-induced hTERT gene inhibition. GM-CSF stimulation inhibited ceramide production, JNK activation, hTERT inhibition, and senescence markers induction. To conclude, our study suggests that, at least in some AML cells, TNFα is a regulator of hTERT with important functional consequences in terms of growth capacity and genetic stability. Moreover, the pro-senescent effect of TNFα is efficiently counter-regulated by stimulatory cytokines such as GM-CSF.

Induction of G1 Arrest and Apoptosis in a Conditional Expression Model of AML1/ETO: P53-Independent Upregulation of P21/WAF/CIP1.

Background: The translocation (8;21) is the most common chromosomal rearrangement in AML, resulting in the expression of the fusion protein AML1/ETO. We have developed an ecdysone-inducible U937 model, in which AML1/ETO is expressed in response to treatment with Ponasterone (Pon) A (Fliegauf et al, Oncogene 2004). This model system was used to determine the cellular effects of AML1/ETO and to identify its target genes in U937 cells.Methods: Effects of AML1/ETO expression upon cell growth, viability, cell cycle and apoptosis were analyzed by trypan blue exclusion, FACS analysis using propidium iodide and DiOC6 staining, DNA laddering and Western blot for PARP cleavage, respectively. The gene expression profile of U937 with and without conditional AML1/ETO expression was assessed using Affymetrix U133A microarrays. Wild-type U937 cells with and without PonA treatment as well as AML1/ETO-negative and AML1/ETO-positive myeloid cell lines served as controls. Northern and Western Blotting were used for validation of expression changes.Results: Induction of AML1/ETO expression in U937 resulted in reduced cell growth, G1 arrest and in apoptosis beginning 48–72 hours after PonA treatment. To investigate the underlying mechanisms, microarray analysis was performed. Expression profiles of AML1/ETO-positive and AML1/ETO-negative cell lines formed distinct clusters. Based on stringent criteria, 191 different genes were found upregulated, whereas 37 were downregulated upon expression of AML1/ETO in U937. The identified genes were screened for genes with known functions in cell cycle and apoptosis by automated and manual review and included 13 apoptosis-related genes. Among them, the CDK inhibitor p21/WAF/CIP1 was upregulated 19-fold upon induction of AML1/ETO, whereas the apoptosis regulator MCL-1 was induced 2.5-fold. Based on our criteria, no differential expression of other transcriptionally-controlled apoptosis regulators (such as BCL2, BAX, BAK1, BAD or c-flip) was noted. Northern and Western Blot analysis confirmed the strong induction of p21/WAF/CIP1 that paralleled the expression of AML1/ETO 10 hours after PonA treatment. Induction of p21/WAF/CIP1 was independent of the tumor suppressor protein p53 (Dou et al., Proc. Natl. Acad. Sci. 1995), and by Western blot, p53 was undetectable in U937. Northern Blot analysis revealed a higher expression of p21/WAF/CIP1 in the AML1/ETO-positive cell lines Kasumi-1 and SKNO-1 than in the AML1/ETO-negative cell lines HL-60, KG-1 and U937, supporting our finding that AML1/ETO may induce p21/WAF/CIP1.Conclusions: AML1/ETO expression resulted in increased expression of p21/WAF/CIP1, which might contribute to the observed growth arrest and induction of apoptosis caused by the conditional expression of AML1/ETO.

AML1/ETO Expresssion in Myeloid Leukemia Cells Is Associated with Enhanced Growth-Inhibitory and P15/INK4b Demethylating Effects of 5-AZA-2′-Deoxycytidine.

The chromosomal translocation (8;21) results in the expression of the chimeric transcription factor AML1/ETO, the most frequent fusion gene in acute myeloid leukemia. The AML1/ETO fusion protein acts as a transcriptional repressor by mediating epigenetic silencing through recruitment of histone deacetylases. Recently, it was shown that it also mediates gene silencing by associating with DNA methyltransferase (Dnmt). We therefore hypothesized that cells expressing AML1/ETO might be preferentially sensitive to the effects of an inhibitor of Dnmt activity, and might provide a superior model for in vitro demethylation and reactivation of the promoter of the p15/INK4b gene (encoding a negative regulator of the cell cycle) that is frequently methylated and silenced in AML and MDS.The 3 myeloid cell lines Kasumi-1 cells (AML1/ETO-positive), KG-1, and KG-1a (both AML1-ETO-negative) are all bearing a heavily methylated p15/INK4b promoter. They were treated with 50 – 1000 nM 5-aza-2′-deoxycytidine (DAC) for three pulses of 24 hrs each. After 6 days, cell growth and viability were determined and FACS analysis performed after propidium iodide staining. Kasumi-1 showed the highest sensitivity to DAC treatment (growth inhibition at 500 nM DAC: Kasumi-1 74.28 %, KG-1 69.16 %, KG-1a 62.38 %). In addition, DAC treatment (500 nM) led to a stronger increase in the sub-G1 fraction in Kasumi-1 (30.46%) compared to KG-1a (20.84 %).Regional p15/INK4b promoter methylation was assessed quantitatively by bisulfite sequencing of ≤10 individual cloned alleles (containing 21 CpGs residues) for calculation of methylated CpG percentage. The p15/INK4b was highly methylated in all 3 cell lines (methylated CpGs Kasumi-1 95.2 %; KG-1 89.6 %; KG-1a 98.4 % ). In Kasumi-1 cells, treatment with DAC resulted in a striking, dose-dependent regional demethylation of the p15/INK4b promoter (demethylated CpGs at 200 nM of DAC: Kasumi-1 63.8 %, KG-1 48.9 %, KG-1a 9.3 %). No demethylating effect was achieved with equitoxic doses of cytarabine or melphalan. Effective demethylation of the p15/INK4b promoter was associated with p15/INK4b protein induction as determined by Western Blot. Simultaneous treatment with all-trans retinoic acid (ATRA) enhanced the effects of DAC treatment upon growth inhibition, but not upon p15/INK4b induction. U937 cells with ecdysone inducible AML1/ETO expression (Fliegauf et al, Oncogene 2004) were also treated with different doses of DAC. When AML1/ETO was induced, U937 cells showed a higher growth inhibition (U937 + AML1/ETO 38.6 %, U937 - AML1/ETO 18 % at 25 nM) and increase in Sub-G1 (U937 + AML1/ETO 18 %, U937 - AML1/ETO 10.55 % at 100 nM) after treatment with DAC.Our results imply that the growth-inhibitory and proapoptotic effect of DAC on leukemia cells is modulated by AML1/ETO protein (or its target genes). The greater accessibility of the p15/INK4b promoter to the demethylating effect of DAC in AML1/ETO expressing Kasumi-1 cells may also be due to differences in regional chromatin structure. With their differential sensitivity to DAC, the cell lines Kasumi-1 and KG-1a provide a model for the different responses of leukemic blasts to DAC.

Identification of Epigenetically Silenced Tumor Suppressor Genes in Myeloid Disorders Leads to the Identification of α-Catenin as a Target Gene in 5q- Syndrome.

CpG islands in the 5′ regulatory regions of genes are generally protected from cytosine methylation as methylation in a promoter can result in transcriptional silencing of the associated gene. Failure of a cell to prevent methylation in the promoter regions of tumor suppressor genes contributes to the onset and progression of cancers. The demethylating agent 5-aza-2′deoxycytidine (DAC) and the histone deacetylase inhibitor suberoyl anilide bishydroxamide (SAHA) possess potent antitumorigenic properties against myeloid disorders. Understanding the alterations of the transcriptome mediated by these drugs should prove vital in uncovering potential tumor suppressor genes epigenetically silenced in myeloid disorders. To this end, we used DAC and SAHA to induce expression of methylated genes in the CD34+ AML cell line KG-1. Expression levels of over 22,000 genes were compared between normal CD34+ cells and treated and untreated KG-1 cells using Affymetrix HG-U133A GeneChip® micoarrays. Statistical analyses revealed 76 genes constitutively expressed in normal CD34+ stem cells, absent in KG-1 cells but whose expression was induced in KG-1 after drug treatment. 39 (51%) of these genes harbored a CpG island in their 5′ regulatory regions, representing potentially methylated tumor suppressor genes in AML. To fit the tumor suppressor paradigm, we hypothesized that any gene possessing antitumorigenic properties would not be expressed in a number of AML patient samples. We examined the expression level of our 39 genes in 120 AML patient samples using microarray analyses. 20 patients belonging to each of the following AML karyotypic groups were analyzed: t(8;21), t(15:17), inv(16), 11q23/MLL, complex and normal karyotpye. Of special note were 8 genes, whose expression was markedly diminished in a subset of patients across all AML karyotypes examined: DAR22, TFIIS, EH-3, ENO2, MXA, DRAL, ASTML and MG50. These represent strong candidates for tumor suppressor genes in AML. Unsupervised clustering analyses using our original 39 genes were performed upon microarray data obtained from patients with myeloproliferative disease (MPD). A subset of 10 genes discriminated between granulocyte samples obtained from healthy donors and those obtained from a subset of agnogenic myeloid metaplasia, essential thronbocythemia and polycythemia vera patients. One of these genes, α-catenin, is located at q31 of chromosome 5, a hot spot for deletion in MDS and AML. α-catenin was expressed in all 120 primary AML samples, including those harboring deletions in chromosome 5. However, Real Time PCR analysis of 32 MDS patients harboring a 5q deletion in the region of α-catenin showed a marked decrease in expression of this gene compared to 20 non 5q- MDS patients. Neighboring genes in the deleted region of 5q did not show as marked a decrease in expression, suggesting loss of expression of both α-catenin alleles in these patients. These findings imply a double hit mechanism in 5q- MDS, where loss of one allele of α-catenin through deletion is supplemented by epigenetic silencing (directly or indirectly) of the second allele. In summary, we have uncovered groups of genes that may be involved in the pathogenesis of AML and various MPDs by virtue of their transcriptional repression through epigenetic events. Importantly, we have identified α-catenin as a key gene on chromosome 5, whose expression is lost in 5q- MDS.

Evidence for Distinct Intracellular Signaling Pathways in CD34+ Progenitor to Dendritic Cell Differentiation from a Human Cell Line Model

Intracellular signals that mediate differentiation of pluripotent hemopoietic progenitors to dendritic cells (DC) are largely undefined. We have previously shown that protein kinase C (PKC) activation (with phorbol ester (PMA) alone) specifically induces differentiation of primary human CD34+ hemopoietic progenitor cells (HPC) to mature DC. We now find that cytokine-driven (granulocyte-macrophage CSF and TNF-alpha) CD34+ HPC-->DC differentiation is preferentially blocked by inhibitors of PKC activation. To further identify intracellular signals and downstream events important in CD34+ HPC-->DC differentiation we have characterized a human leukemic cell line model of this process. The CD34+ myelomonocytic cell line KG1 differentiates into dendritic-like cells in response to granulocyte-macrophage CSF plus TNF-alpha, or PMA (with or without the calcium ionophore ionomycin, or TNF-alpha), with different stimuli mediating different aspects of the process. Phenotypic DC characteristics of KG1 dendritic-like cells include morphology (loosely adherent cells with long neurite processes), MHC I+/MHC IIbright/CD83+/CD86+/CD14- surface Ag expression, and RelB and DC-CK1 gene expression. Functional DC characteristics include fluid phase macromolecule uptake (FITC-dextran) and activation of resting T cells. Comparison of KG1 to the PMA-unresponsive subline KG1a reveals differences in expression of TNF receptors 1 and 2; PKC isoforms alpha, beta I, beta II, and mu; and RelB, suggesting that these components/pathways are important for DC differentiation. Together, these findings demonstrate that cytokine or phorbol ester stimulation of KG1 is a model of human CD34+ HPC to DC differentiation and suggest that specific intracellular signaling pathways mediate specific events in DC lineage commitment.