Expression In Acute Myeloid Leukemia Cells Research Articles

MicroRNA-193a represses c-kit expression and functions as a methylation-silenced tumor suppressor in acute myeloid leukemia

Aberrant activation of c-kit proto-oncogene contributes to abnormal cell proliferation by altering the tyrosine kinase signaling and constitutes a crucial impetus for leukemogenesis. Epigenetic silencing of tumor-suppressive microRNAs (miRNAs) is a key oncogenic mechanism for the activation of oncogenes in tumors. In this study, several miRNAs potentially binding to the 3'-untranslated region of human c-kit mRNA were screened by luciferase reporter assays. Among these miRNAs, miR-193a was embedded in a CpG island and epigenetically repressed by promoter hypermethylation in acute myeloid leukemia (AML) cell lines and primary AML blasts, but not in normal bone marrow cells. Importantly, miR-193a levels were inversely correlated with c-kit levels measured in 9 leukemia cell lines and 27 primary AML samples. Restoring miR-193a expression in AML cells harboring c-kit mutation and/or overexpression, either by synthetic miR-193a transfection or by DNA hypomethylating agent 5-azacytidine (5-aza) treatment, resulted in a significant reduction in c-kit expression at both RNA and protein levels and inhibition of cell growth. The growth-inhibitory activity of miR-193a was associated with apoptosis and granulocytic differentiation. Moreover, 5-aza-induced c-kit reduction could be partially blocked by miR-193a inhibitor, leading to a reversal of antiproliferative and proapoptotic effects of 5-aza. These data reveal a critical role for methylation-repressed miR-193a in myeloid leukemogenesis and the therapeutic promise of upregulating miR-193a expression for c-kit-positive AML.

The FOXM1 transcriptional factor promotes the proliferation of leukemia cells through modulation of cell cycle progression in acute myeloid leukemia

FOXM1 is an important cell cycle regulator and regulates cell proliferation. In addition, FOXM1 has been reported to contribute to oncogenesis in various cancers. However, it is not clearly understood how FOXM1 contributes to acute myeloid leukemia (AML) cell proliferation. In this study, we investigated the cellular and molecular function of FOXM1 in AML cells. The FOXM1 messenger RNA (mRNA) expressed in AML cell lines was predominantly the FOXM1B isoform, and its levels were significantly higher than in normal high aldehyde dehydrogenase activity (ALDH(hi)) cells. Reduction of FOXM1 expression in AML cells inhibited cell proliferation compared with control cells, through induction of G(2)/M cell cycle arrest, a decrease in the protein expression of Aurora kinase B, Survivin, Cyclin B1, S-phase kinase-associated protein 2 and Cdc25B and an increase in the protein expression of p21(Cip1) and p27(Kip1). FOXM1 messenger RNA (mRNA) was overexpressed in all 127 AML clinical specimens tested (n = 21, 56, 32 and 18 for M1, M2, M4 and M5 subtypes, respectively). Compared with normal ALDH(hi) cells, FOXM1 gene expression was 1.65- to 2.26-fold higher in AML cells. Moreover, the FOXM1 protein was more strongly expressed in AML-derived ALDH(hi) cells compared with normal ALDH(hi) cells. In addition, depletion of FOXM1 reduced colony formation of AML-derived ALDH(hi) cells due to inhibition of Cdc25B and Cyclin B1 expression. In summary, we found that FOXM1B mRNA is predominantly expressed in AML cells and that aberrant expression of FOXM1 induces AML cell proliferation through modulation of cell cycle progression. Thus, inhibition of FOXM1 expression represents an attractive target for AML therapy.

Expression of heme oxygenase‐1 in human leukemic cells and its regulation by transcriptional repressor Bach1

Heme oxygenase (HO)-1 has anti-oxidative, anti-inflammatory, and anti-apoptotic activities. However, little is known about the regulation of HO-1 in human primary acute myeloid leukemia (AML) cells. Here we investigated the expression of HO-1 in primary and established AML cells as well as other types of leukemic cells and normal monocytes, and its regulatory mechanism by the transcriptional repressor, BTB and CNC homology 1 (Bach1), and the activator, nuclear factor erythroid-derived 2 related factor 2 (Nrf2). Leukemic cell lines such as U937 expressed little HO-1, whereas most freshly isolated AML cells and monocytes expressed substantial amounts of HO-1, along with Bach1 and Nrf2. When U937 cells were treated with phorbol myristate acetate (PHA) or gamma-interferon, they significantly expressed both HO-1 and Bach1, like primary AML cells. Treatment with lipopolysaccharide (LPS) enhanced HO-1 expression in U937 cells but suppressed it in primary monocytes and PMA-treated U937 cells. In HO-1-expressing cells, Bach1 was localized in the cytoplasm, but Nrf2 was localized in the nuclei. Chromatin immunoprecipitation assay of these cells revealed the preferential binding of Nrf2 over Bach1 to Maf-recognition elements, the enhancer regions of the HO-1 gene. The downregulation of the HO-1 gene with siRNA increased a cytotoxic effect of an anticancer drug on primary AML cells, whereas the downregulation of Bach1 increased HO-1 expression, leading to enhanced survival. These and other results show that Bach1 plays a critical role in regulating HO-1 gene expression in AML cells and its expression suppresses their survival by downregulating HO-1 expression. Thus, functional upregulation of Bach1 is a potential strategy for antileukemic therapy.

Signatures of polycomb repression and reduced H3K4 trimethylation are associated with p15INK4b DNA methylation in AML

AbstractDNA hypermethylation of the p15INK4b tumor suppressor gene is commonly observed in acute myeloid leukemia (AML). Repressive histone modifications and their associated binding proteins have been implicated in the regulation of DNA methylation and the transcriptional repression of genes with DNA methylation. We have used high-density chromatin immunoprecipitation-on-chip to determine the histone modifications that normally regulate p15INK4b expression in AML cells and how these marks are altered in cells that have p15INK4b DNA methylation. In AML patient blasts without p15INK4b DNA methylation, a bivalent pattern of active (H3K4me3) and repressive (H3K27me3) modifications exist at the p15INK4b promoter. AML patient blasts with p15INK4b DNA methylation lose H3K4me3 at p15INK4b and become exclusively marked by H3K27me3. H3K27me3, as well as EZH2, extends throughout p14ARF and p16INK4a, indicating that polycomb repression of p15INK4b is a common feature in all AML blasts irrespective of the DNA methylation status of the gene. Reactivation of p15INK4b expression in AML cell lines and patient blasts using 5-aza-2′-deoxycytidine (decitabine) and trichostatin A increased H3K4me3 and maintained H3K27me3 enrichment at p15INK4b. These data indicate that AML cells with p15INK4b DNA methylation have an altered histone methylation pattern compared with unmethylated samples and that these changes are reversible by epigenetic drugs.

Expression patterns of leptin receptor (OB-R) isoforms and direct in vitro effects of recombinant leptin on OB-R, leptin expression and cytokine secretion by human hematopoietic malignant cells

Several studies have implicated leptin in the pathophysiology of neoplasias. We investigated the direct effect of leptin on malignant hematopoietic tissue that included: primary acute myeloid leukemia (AML) cells, leukemic cell lines and bone marrow biopsies from multiple myeloma (MM) patients. PBMC, T-cells, B-cells and monocytes from healthy subjects served as controls. We defined the patterns of OB-R isoform expression in AML cells and leukemic cell lines in comparison to control cells by RT-PCR. rLeptin upregulated the expression of OB-R and endogenous leptin in AML blasts and certain cell lines but not in control cells. Cytometric Bead Array analysis of pro- and anti-inflammatory cytokines showed that rleptin upregulates IL-6 secretion by AML cells, various cytokines by the leukemic cell lines tested and IL-10 secretion by control PBMC, contributed by monocytes. Western immunoblotting revealed that the effect of rleptin was independent of JAK-2/phospho-JAK-2 protein levels. Finally, MM biopsies stained positive for leptin and, to a lesser extend, OB-R. Immunoreactivity was confined mostly to the nucleus of the myeloma cells. Normal myelocytes, promyelocytes and megakaryocytes stained weakly positive, and erythroid cells were constantly negative. We propose that the leptin/OB-R system is strongly and directly involved in supporting the growth of hematopoietic malignancies.

Significance of Notch Expression in Acute Myeloid Leukemia

Background: Notch is a gene family encoding receptors to transduce intercellular signals involved in cell-fate determination. Although several lines of evidence indicate that abnormal Notch signaling may contribute to neoplastic transformation, little is known regarding the role of Notch in the pathogenesis of leukemia. Methods: To explore the functional significance of Notch1 in acute myeloid leukemia (AML), the expression of Notch1 and its association with survivin and p27Kip1 expression was examined in 50 patients with de novo AML. Results: Notch1 transcripts were expressed in 40 (80%) cases with a variable degree of expression, and the fraction of AML cells in the G0/G1 phase was higher in Notch1-positive cases than in Notch1-negative cases. Survivin was shown to be present in 38 (76.0%) cases, and Notch1 expression highly correlated with survivin mRNA expression (r=0.7170, P＜0.001). p27Kip1 was present in 40 (80.0%) cases of AML and p27Kip1 expression was significantly associated with Notch1 expression (r=0.8770, P＜0.001). Except for one case, the simultaneous expression of survivin and p27Kip1 was not seen in all cases that were negative for Notch1 expression. There were no differences in clinical outcomes according to Notch1 expression. Conclusion: Notch1 expression was a frequent event and has functional significance in the alteration of the cell cycle in AML cells. Notch1 expression was also significantly associated with survivin and p27kip1 expression in AML cells. To evaluate the clinical significanceand functional role of Notch1 expression in the aberrant regulation of survivin and p27Kip1 expression in de novo AML, a further study with a larger number of patients is necessary.

Toll-Like Receptor Agonists Induce Immunogeneicity and Apoptosis of Acute Myeloid Leukemia Cells.

Acute myeloid leukemia (AML) is a common form of acute leukemia and remains a difficult disease with poor survival in patients who have failed standard therapy. New therapeutic strategies are needed to achieve longer survival and improve cure rates in AML patients. Toll-like receptor (TLR) agonists have been shown to elicit anti-leukemia effects in murine AML models. However, TLR expression profile of human AML cells is unknown. We analyzed TLR1-10 mRNA expression in purified AML cells from 41 patients with different AML subtypes (M0, M1, M2, M3, M4, or M5; n > 5 per group) by real-time RT-PCR. The majority of AML samples expressed high level of TLR2, 4, 7, 8, low level of TLR1, 5, 9, 10, and undetectable level of TLR3. Significant higher TLR4 and TLR7 expressions were detected on M4 and M5 subtypes of AML cells. Triggering TLR4 or TLR7 with specific TLR agonists (Monophosphoryl Lipid A or Imiquimod) significantly increased the surface expression of molecules essential for T cell activation (CD54, CD80, CD86) on AML M4/M5 cells and enhanced T-cell mediated proliferative responses against AML cells. Thus, TLR signaling enhances the immunogenicity of AML M4/M5 cells and makes them more suitable targets for T cell mediated attack. Most importantly, TLR7 agonist strongly induced apoptotic death of primary AML M4/M5 cells and inhibited the growth of TLR7-expressing AML cell lines (U937, HL-60, KG-1) in culture in a drug dose dependent manner. The addition of TLR7 agonist at 10 ug/ml fully induced apoptosis of AML cells and inhibited the growth of AML cell lines, as confirmed by viable cell counts and TMRE staining. Intracellular staining demonstrated that TLR7 agonist treatment significantly down-regulated the signal transducer and activator of transcription (STAT)3 and STAT5 protein expression in AML cells. These results suggest that TLR7 agonist-induced apoptosis of AML cells is likely via inhibition of STAT3 and/or STAT5 signaling pathway. To study the in vivo effects of TLR7 agonist against human AML cells, primary AML M4/M5 cells or a monocytic AML cell line (U937) were injected i.p. into NOD-SCID IL2Rgamma<null> mice with or without subsequent TLR7 agonist treatment. Mice receiving TLR7 agonist treatment (1 mg/kg daily i.p. infusion for 5 days) significantly reduced tumor burden with substantially lower numbers of engrafted leukemia cells detected in these xenograft mice. Flow cytometry results confirmed that residual AML cells recovered from mice treated with TLR7 agonist were apoptotic with down-regulated expression of TMRE and STAT3/STAT5, confirming previous in vitro findings that TLR7 agonist-treated AML cells are programmed to die. The antitumor efficacy of systemic administration of TLR7 agonist in NOD/SCID mice with established human AML is being investigated using these xenograft mouse models. In summary, our results provide the first report of TLR expression profile of human AML cells and demonstrate that TLR targeting of AML cells can increase the immunogeneicity of leukemia cells and directly induce AML apoptosis in vitro and in vivo, providing new insights into the biology and therapy of AML.

Bmi-1 Expression Is Useful To Design Therapy and To Predict Prognosis in Patients with Acute Myeloid Leukemia.

Since prognosis of patients with acute myeloid leukemia (AML) is highly variable even in a single subpopulation in FAB classification, it would be useful to find prognostic molecular markers for AML. Thus, we investigated Bmi-1 expression in AML cells by flow cytometry and analyzed whether it predicts prognosis in AML patients and further it is helpful to choose therapies in the modalities of treatment options, because it is known to be required for self-renewal mechanism of leukemic stem cells. Bmi-1 expression in bone marrow or peripheral blood cells was analyzed in 49 patients with AML (M0(n=5), M1(n=7), M2(n=6), M3(n=5), M4(n=8), M5(n=5), M6(n=1)), granulocytic sarcoma(n=1), MDS-AML (n=9), and secondary AML(n=2). Freshly isolated AML cells were stained with a PE-conjugated anti-CD34-antibody followed by fixation and then with anti-Bmi-1-antibody-FITC. All of patients with low Bmi-1 positivity (<35%, n=11) except for de novo AML(M0) entered in complete remission (CR) with single induction chemotherapy(n=5) and accordingly had better overall survival, even though lower dose of chemotherapy (60% of standard dose) was given (n=3). Alternatively, patients with higher percentage of Bmi-1 expression in AML cells (> 70%, n=19) except for AML(M3) progressed to death within two years, unless they were treated with highly intensive therapy such as high dose AraC or allogeneic stem cell transplantation (n=3). Patients with intermediate degree of Bmi-1 expression (35–60%, n=5) responded to standard intensity of chemotherapy (n=2) and are alive for more than two years. Interestingly, patients with MDS-AML (n=9) had high Bmi-1 expression (79%) and all of them have died within 20 months. Binary logistic regression model showed that significant correlation was found among survival status as dependent variable, Bmi-1, and treatment intensity as independent variable (p = 0.004). On the other hand, Univariate analysis did not reveal any relation of Bmi-1 expression to karyotype, age, WBC count, or FAB subtype. In conclusion, Bmi-1 expression could be an independent prognostic marker and useful tool to design therapy for the AML patients.

Decitabine: Where Is the Target?.

The methylation inhibitor 5-Aza-2′-deoxycytidine (decitabine) has therapeutic efficacy in acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS). Using microarray analysis, we investigated global changes in gene expression after decitabine treatment in AML. In the AML cell line OCI-AML2, decitabine induced the expression of 81 out of 22 000 genes; 96 genes were downregulated (X2-fold change in expression). RT-PCR analysis of 10 randomly selected genes confirmed the changes of expression in AML cells. Similar results were obtained with primary AML and MDS cells after treatment with decitabine ex vivo and in vivo, respectively. In contrast, significantly fewer changes in gene expression and cytotoxicity were detected in normal peripheral blood mononuclear and bone marrow cells or transformed epithelial cells treated with decitabine. Interestingly, only 50.6% of the induced genes contain putative CpG islands in the 5′ region. To further investigate the significance of promoter methylation in the induced genes, we analyzed the actual methylation status of randomly selected decitabine-inducible genes. We detected hypermethylation exclusively in the 5′ region of the myeloperoxidase (MPO) gene. DNA methylation inversely correlated with MPO expression in newly diagnosed untreated AML patients (P<0.004). In contrast, all other analyzed decitabine-inducible genes revealed no CpG methylation in the promoter region, suggesting a methylation-independent effect of decitabine.

The value of the MDR1 reversal agent PSC-833 in addition to daunorubicin and cytarabine in the treatment of elderly patients with previously untreated acute myeloid leukemia (AML), in relation to MDR1 status at diagnosis

AbstractTo determine whether MDR1 reversal by the addition of the P-glycoprotein (P-gp) inhibitor PSC-833 to standard induction chemotherapy would improve event-free survival (EFS), 419 untreated patients with acute myeloid leukemia (AML) aged 60 years and older were randomized to receive 2 induction cycles of daunorubicin and cytarabine with or without PSC-833. Patients in complete remission were then given 1 consolidation cycle without PSC-833. Neither complete response (CR) rate (54% versus 48%; P = .22), 5-year EFS (7% versus 8%; P = .53), disease-free survival (DFS; 13% versus 17%; P = .06) nor overall survival (OS; 10% in both arms; P = .52) were significantly improved in the PSC-833 arm. An integrated P-gp score (IPS) was determined based on P-gp function and P-gp expression in AML cells obtained prior to treatment. A higher IPS was associated with a significantly lower CR rate and worse EFS and OS. There was no significant interaction between IPS and treatment arm with respect to CR rate and survival, indicating also a lack of benefit of PSC-833 in P-gp–positive patients. The role of strategies aimed at inhibitory P-gp and other drug-resistance mechanisms continues to be defined in the treatment of patients with AML.

FLT3/ITD Mutation Signaling Includes Suppression of SHP-1

Mutations in the FLT3 gene are the most common genetic alteration found in AML patients. FLT3 internal tandem duplication (ITD) mutations result in constitutive activation of FLT3 tyrosine kinase activity. The consequences of this activation are an increase in total phosphotyrosine content, persistent downstream signaling, and ultimately transformation of hematopoietic cells to factor-independent growth. The Src homology (SH)2 domain-containing protein-tyrosine phosphatase (SHP)-1 is involved in the down-regulation of a broad range of growth factor and cytokine-driven signaling cascades. Loss-of-function or deficiency of SHP-1 activity results in a hyperproliferative response of myelomonocytic cell populations to growth factor stimulation. In this study, we examined the possible role of SHP-1 in regulating FLT3 signaling. We found that transformation of TF-1 cells with FLT3/ITD mutations suppressed the activity of SHP-1 by approximately 3-fold. Suppression was caused by decreased SHP-1 protein expression, as analyzed at both the protein and RNA levels. In contrast, protein levels of SHP-2, a phosphatase that plays a stimulatory role in signaling through a variety of receptors, did not change significantly in FLT3 mutant cells. Suppressed SHP-1 protein levels in TF-1/ITD cells were partially overcome after cells were exposed to CEP-701, a selective FLT3 inhibitor. SHP-1 protein levels also increased in naturally occurring FLT3/ITD expressing AML cell lines and in primary FLT3/ITD AML samples after CEP-701 treatment. Furthermore, a small but reproducible growth/survival advantage was observed in both TF-1 and TF-1/ITD cells when SHP-1 expression was knocked down by RNAi. Taken together, these data provide the first evidence that suppression of SHP-1 by FLT3/ITD signaling may be another mechanism contributing to the transformation by FLT3/ITD mutations.

The Anti-Proliferative Effect of Immunotoxin Gemtuzimab Ozogamycin (GO) (Mylotarg) to Acute Myeloid Leukemia (AML) Cells Is Associated with the Level of Protein Kinase Syk Exspression.

AML cells express the cell surface antigen CD33 that serves as a down-regulator of cell growth. Anti-CD33 monoclonal antibody (mAb) coupled to a toxin is a licensed drug for the treatment of relapsed AML (Mylotarg). Syk is not only an essential element in several cascades coupling antigen receptors to cell responses, but also SYK is a tumor suppressor gene. Silencing of SYK by hypermethylation results in absence of Syk expression and unresponsiveness of tumor cells to various treatments.. Earlier we demonstrated that about 30% of the AML samples were Syk-negative and the response of leukemia cells to CD33 ligation correlated with Syk expression. Therefore, here we investigated whether or not the response of the AML cells to GO treatment also depends on the level of Syk expression. It is even more intriguing since 50% of Mylotarg is not conjugated to calicheamicin, and hence some of its activity is in fact due to anti-CD33 mAb signaling. Primary leukemia cells were analyzed for their level of the Syk expression (11 Syk positive and 6 Syk-negative) and then treated with GO. In both groups GO mediated dose-dependent inhibition of proliferation, however the level of inhibition in Syk-positive group was significantly higher (p<0.003). The largest differences were notices at low doses (1–50 ng/ml) of GO, while at higher doses (>100 ng/ml) this effect was diminished. Moreover, 8 of 11 (72.7%) samples in Syk-positive group were “good” responders to GO (inhibition>50%), while in Syk-negative group only 1 of 6 (16.6%) samples demonstrated similar response. This difference was statistically significant and suggesting a correlation between the level of Syk expression in AML cells and their response to Mylotarg. After treatment of Syk-negative AML samples with methylation inhibitor 5-aza-cytidine in 2 of 6 samples Syk protein expression was restored. However, in all 6 samples after 5-aza-CdR treatment GO inhibitory activity was significantly higher (p<0.005). Moreover, after 5-aza-cytidine treatment, these initially CD33 ligation non-responsive cells demonstrated high level (up to 70%) inhibition of proliferation upon anti-CD33 mAb treatment. Analysis of CD33 ligation mediated signaling in 3 Syk-positive samples (1 responsive and 2 unresponsive to GO treatment) revealed significant differences: in responsive AML sample Syk became phosphorylated and created complexes with CD33 and protein phosphatase SHP-1, while in non-responsive samples were no Syk/CD33 or Syk/SHP-1 complexes. This does imply that not only the physical presence of Syk, but also its functional activity is important for the adequate response of AML cells to Mylotarg. To create a model with down-regulated Syk we treated Syk-positive human AML cell lines with various doses of the Syk inhibitor Piceatannol and analyzed their proliferative response to Mylotarg. In AML cell lines treated with 10–100 μM of Piceatannol, GO inhibitory activity was significantly lower compared to untreated cells or even those treated by low doses of Piceatannol. This confirmed our observation that expression and functional activity of Syk is associated with the ability of AML cells to respond to GO treatment. We hypothesize that the pre-treatment level of Syk expression may be used as a prognostic factor for response to Mylotarg treatment. Screening for Syk expression in leukemia cell samples from patients before treatment might allow rational selection of patients for this expensive and toxic therapy and thus improve its success rate in the clinic.

EB10, an Anti-FLT3 Monoclonal Antibody, Prolongs Survival and Reduces NOD/SCID Engraftment of AML Cell Lines and Primary Blasts.

The FLT3 receptor is a potential target in AML due to its role in leukemogenesis and its high degree of expression on blasts from approximately 90% of acute myeloid leukemia (AML) patients. In addition, mutant forms of FLT3, including internal tandem duplications (ITD) in the juxtamembrane region and point mutations in the kinase domain, constitutively activate FLT3 signaling. ITD mutations in particular are also associated with poor prognosis. A number of small molecule tyrosine kinase inhibitors (TKI) against FLT3 are currently in clinical trials and have shown some clinical activity. However, TKIs have various limitations, including their lack of specificity, which may produce toxicities, and can select for drug resistant cells. In an attempt to overcome some of these limitations and to generate new agents which might cooperate in targeting FLT3, we generated a fully humanized phage display monoclonal antibody (EB10). This antibody is capable of inhibiting both ligand-activated wild-type and, to a lesser degree, ligand-independent mutant FLT3 signaling. When EB10 is used to treat cells expressing activated FLT3, inhibition of downstream pathways including STAT5, AKT and MAPK are also frequently seen. EB10 treatment of cells expressing FLT3 in the presence of NK cells leads to antibody-dependent cell-mediated cytotoxicity (ADCC). EB10 treatment of NOD/SCID mice injected with FLT3 expressing AML cell lines or with primary AML blasts significantly prolongs survival and/or reduces engraftment of leukemic cells. EB10 proved efficacious in vivo against cells even when in vitro EB10 treatment did not significantly reduce FLT3 signaling. This indicates that ADCC may be the primary mechanism mediating cytotoxicity as opposed to direct FLT3 inhibition. In contrast to the effects on AML cell lines and primary samples, EB10 treatment did not significantly reduce NOD/SCID engraftment of normal human CD34+ hematopoietic stem cells. Anti-FLT3 antibodies, like EB10, may be a promising therapeutic agent that can specifically target malignant cells with limited toxicities against normal hematopoietic stem cells and should be considered for clinical trials.

Induction of gene expression by 5-Aza-2′-deoxycytidine in acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS) but not epithelial cells by DNA-methylation-dependent and -independent mechanisms

The methylation inhibitor 5-Aza-2'-deoxycytidine (5-Aza-CdR, decitabine) has therapeutic efficacy in acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS). Using microarray analysis, we investigated global changes in gene expression after 5-Aza-CdR treatment in AML. In the AML cell line OCI-AML2, Aza-CdR induced the expression of 81 out of 22 000 genes; 96 genes were downregulated (> or =2-fold change in expression). RT-PCR analysis of 10 randomly selected genes confirmed the changes of expression in AML cells. Similar results were obtained with primary AML and MDS cells after treatment with 5-Aza-CdR ex vivo and in vivo, respectively. In contrast, significantly fewer changes in gene expression and cytotoxicity were detected in normal peripheral blood mononuclear and bone marrow cells or transformed epithelial cells treated with 5-Aza-CdR. Interestingly, only 50.6% of the induced genes contain putative CpG islands in the 5' region. To further investigate the significance of promoter methylation in the induced genes, we analyzed the actual methylation status of randomly selected 5-Aza-CdR-inducible genes. We detected hypermethylation exclusively in the 5' region of the myeloperoxidase (MPO) gene. DNA methylation inversely correlated with MPO expression in newly diagnosed untreated AML patients (P< or =0.004). In contrast, all other analyzed 5-Aza-CdR-inducible genes revealed no CpG methylation in the promoter region, suggesting a methylation-independent effect of 5-Aza-CdR.

Stem cell factor as a survival and growth factor in human normal and malignant hematopoiesis.

Stem cell factor (SCF) is an essential hematopoietic cytokine that interacts with other cytokines to preserve the viability of hematopoietic stem and progenitor cells, to influence their entry into the cell cycle and to facilitate their proliferation and differentiation. SCF on its own cannot drive noncycling hematopoietic progenitor cells into the cell cycle but does prevent their apoptotic death. SCF when combined with other cytokines increases the cloning efficacy of hematopoietic progenitor cells from all lineages. SCF also stimulates the growth of CD34+ leukemic progenitor cells from most patients with acute myeloid leukemia (AML). The mRNA expression of the SCF receptor c-kit has been shown to be significantly increased in all fresh AML blast cells compared with normal controls (healthy volunteers), in particular CD34+ cells. Two inhibitory cytokines, transforming growth factor-beta and interleukin-4, decreased c-kit expression, whereas tumor necrosis factor-alpha increased c-kit expression, but chemotherapeutic drugs showed no effect on c-kit expression, but chemotherapeutic drugs showed no effect on c-kit expression in AML cells. Apoptosis has been shown to be directly related to a high complete remission rate in AML patients following induction therapy. Since SCF has been shown to stimulate the proliferation of mainly CD34+ AML cells, we have investigated whether the poor response of patients with CD34+ myeloid leukemia cells to chemotherapy could be due to SCF-induced resistance to apoptosis. The effect of SCF on the apoptosis induced by chemotherapeutic drugs commonly used in the treatment of AML - cytarabine, daunorubicin and carboplatin - was examined in human CD34+ myeloid leukemia cells in serum-free cultures. SCF significantly reduced the induced apoptosis by more than 50% in all CD34+ human leukemia cells treated by any of the three chemotherapeutic drugs. Antibodies blocking c-kit reversed the significant inhibitory effect of SCF on chemotherapy-induced apoptosis, confirming the role of SCF in the resistance to chemotherapy-induced apoptosis in CD34+ human leukemia. These results suggest that the poor response of patients with CD34+ leukemia cells could be at least partially due to less chemotherapy-induced apoptosis resulting from protection by SCF as an adjuvant mechanism for drug resistance in myeloid leukemia. We conclude that an antisense strategy to block c-kit expression in AML blast cells may prove valuable for decreasing the chemoresistance of AML patients. The abrogation of leukemic resistance to apoptotic death through anti-SCF/c-pit expression combined with chemotherapy offers potential for designing novel therapeutic approaches for refractory AML patients.