cytochrome-c-t and Myelodysplastic-Syndromes

The Myelodysplastic Syndromes are stem cell heterogeneous disorders characterized by peripheral cytopenias and hypercellular bone marrow, which can evolute to acute leukaemia. Vitamin C can act as an antioxidant, ascorbic acid (AA) donates two electrons and becomes oxidized to dehydroascorbic acid (DHA). Under physiological conditions, vitamin C predominantly exists in its reduced (AA) form but also exists in trace quantities in the oxidized form (DHA). This study evaluates the therapeutic potential of vitamin C in Myelodysplastic Syndromes (MDSs). F36P cells (MDS cell line) were treated with ascorbate and dehydroascorbate alone and in combination with cytarabine. Cell proliferation and viability were assessed by trypan blue assay and cell death was evaluated by optical microscopy and flow cytometry. The role of reactive oxygen species, mitochondrial membrane potential, BAX, BCL-2 and cytochrome C were also assessed. Vitamin C decreases cell proliferation and viability in a concentration, time and administration dependent-manner inducing cell death by apoptosis, which was shown to be associated to an increased in superoxide production, mitochondrial membrane depolarization. These compounds modulate BCL-2, BAX and cytochrome C release. These results suggest that vitamin C induces cell death trough apoptosis in F36P cells and may be a new therapeutic approach in Myelodysplasia.

Topics: Apoptosis; Ascorbic Acid; bcl-2-Associated X Protein; Catalase; Cell Line; Cell Proliferation; Cell Survival; Cytochromes c; Dehydroascorbic Acid; Humans; Membrane Potential, Mitochondrial; Myelodysplastic Syndromes; Reactive Oxygen Species; Superoxide Dismutase

Myelodysplastic syndrome (MDS) is a preneoplastic condition that frequently develops into overt acute myeloid leukemia (AML). The P39 MDS/AML cell line manifested constitutive NF-kappaB activation. In this cell line, NF-kappaB inhibition by small interfering RNAs specific for p65 or chemical inhibitors including bortezomib resulted in the down-regulation of apoptosis-inhibitory NF-kappaB target genes and subsequent cell death accompanied by loss of mitochondrial transmembrane potential as well as by the mitochondrial release of the caspase activator cytochrome c and the caspase-independent death effectors endonuclease G and apoptosis-inducing factor (AIF). Bone marrow cells from high-risk MDS patients also exhibited constitutive NF-kappaB activation similar to bone marrow samples from MDS/AML patients. Purified hematopoietic stem cells (CD34+) and immature myeloid cells (CD33+) from high-risk MDS patients demonstrated the nuclear translocation of the p65 NF-kappaB subunit. The frequency of cells with nuclear p65 correlated with blast counts, apoptosis suppression, and disease progression. NF-kappaB activation was confined to those cells that carried MDS-associated cytogenetic alterations. Since NF-kappaB inhibition induced rapid apoptosis of bone marrow cells from high-risk MDS patients, we postulate that NF-kappaB activation is responsible for the progressive suppression of apoptosis affecting differentiating MDS cells and thus contributes to malignant transformation. NF-kappaB inhibition may constitute a novel therapeutic strategy if apoptosis induction of MDS stem cells is the goal.

Topics: Active Transport, Cell Nucleus; Apoptosis; Apoptosis Inducing Factor; Bone Marrow Cells; Case-Control Studies; Cell Differentiation; Cell Line, Tumor; Cell Nucleus; Cell Transformation, Neoplastic; Chromosome Aberrations; Cytochromes c; Endodeoxyribonucleases; Humans; Mitochondria; Myelodysplastic Syndromes; Myeloid Progenitor Cells; Risk Factors; RNA, Small Interfering; Transcription Factor RelA

The DNA methylation inhibitor 5-Aza-2'-deoxycytidine (5-Aza-CdR) has significant therapeutic value for the treatment of patients with myelodysplastic syndrome (MDS), acute myeloid leukemia (AML) and chronic myeloid leukemia (CML). The demethylating effect of 5-Aza-CdR has been well characterized. In contrast, less is known about the molecular events downstream of the methylation inhibition. Here, 5-Aza-CdR induced apoptosis in AML cells (both p53 mutant and wild-type) but not in epithelial or normal PBMCs. Cell death was accompanied by activation of the mitochondrial apoptosis pathway, as shown by release of cytochrome c and AIF and loss of mitochondrial membrane potential (DeltaPsim). Activation of caspase-3 (but not -6 and -8) was detectable using Western blot analysis and measurement of caspase enzymatic activity. 5-Aza-CdR treatment resulted in the induction of p21, which correlated with the arrest of AML cells in the G1 cell cycle phase. Induction of p21 expression was independent of its promoter methylation status but mediated by 5-Aza-CdR-induced reexpression of the tumor-suppressor p73, a known upstream regulator of p21. The p73 promoter was hypermethylated in AML cell lines and in primary AML cells but not in epithelial cells, which were resistant toward 5-Aza-CdR. Therefore, 5-Aza-CdR-mediated specific killing of myeloid cells might be dependent on its ability to revert p73 promoter methylation and to reexpress p73 mRNA. In addition, exogenous expression of p73 rendered epithelial cells sensitive to apoptosis induced by 5-Aza-CdR or other cytostatic drugs. We therefore conclude that p73 is a relevant target for methylation-dependent efficacy of 5-Aza-CdR in AML cells.

Topics: Apoptosis; Azacitidine; Blotting, Western; Caspase 3; Caspases; Cell Cycle; Cell Cycle Proteins; Cell Line, Tumor; CpG Islands; Cyclin-Dependent Kinase Inhibitor p21; Cytochromes c; Decitabine; DNA Methylation; DNA-Binding Proteins; Epithelial Cells; Flow Cytometry; G1 Phase; Genes, Tumor Suppressor; HeLa Cells; HL-60 Cells; Humans; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Leukemia, Myeloid, Acute; Leukocytes, Mononuclear; Membrane Potentials; Microscopy, Fluorescence; Mitochondria; Myelodysplastic Syndromes; Nuclear Proteins; Promoter Regions, Genetic; Reverse Transcriptase Polymerase Chain Reaction; RNA; RNA, Messenger; Sulfites; Time Factors; Transfection; Tumor Protein p73; Tumor Suppressor Protein p53; Tumor Suppressor Proteins; U937 Cells; Up-Regulation

Early erythroblasts from patients with refractory anemia (RA) and RA with ringed sideroblasts (RARS) show constitutive mitochondrial release of cytochrome c. Moreover, mature erythroblasts in RARS, but not in RA, display aberrant accumulation of mitochondrial ferritin (MtF). We analyzed cytochrome c release, MtF expression, and gene expression during erythroid differentiation in bone marrow cells from myelodysplastic syndrome (MDS) patients and healthy controls. Whereas none or few cultured erythroid cells from healthy individuals and RA patients expressed MtF, those from RARS patients showed MtF expression at an early stage, when cells were CD34+ and without morphologic signs of erythroid differentiation. The proportion of RARS erythroblasts that were MtF+ increased further upon in vitro maturation. Moreover, a significant overexpression of mRNA encoding cytochrome c, and proapoptotic Bid and Bax, was seen in freshly isolated cells from MDS patients. Genes involved in erythroid differentiation were also dysregulated in MDS cells. Importantly, GATA-1 expression increased during normal erythroid maturation, but remained low in MDS cultures, indicating a block of erythroid maturation at the transcriptional level. In conclusion, aberrant MtF expression in RARS erythroblasts occurs at a very early stage of erythroid differentiation and is paralleled by an up-regulation of genes involved in this process.

Topics: Adenosine Triphosphate; Anemia, Sideroblastic; Apoptosis; bcl-2-Associated X Protein; BH3 Interacting Domain Death Agonist Protein; Carrier Proteins; Cytochromes c; DNA-Binding Proteins; Erythroblasts; Erythroid-Specific DNA-Binding Factors; Ferritins; GATA1 Transcription Factor; Gene Expression; Glycoproteins; Granulocyte Colony-Stimulating Factor; Hematopoietic Stem Cells; Hemoglobins; Humans; Iron; Mitochondria; Myelodysplastic Syndromes; Proto-Oncogene Proteins c-bcl-2; Risk Factors; RNA, Messenger; Transcription Factors