cyclin-d1 and Myelodysplastic-Syndromes

We previously demonstrated upregulation of c-myc, survivin, and cyclin D1 in CD34+ bone marrow mononuclear cells (BMMNCs) of patients with trisomy 8 and monosomy 7 myelodysplastic syndromes (MDS). "Knockdown" of cyclin D1 by RNA interference decreased trisomy 8 cell growth, suggesting that this might be a therapeutic target in MDS.. We performed preclinical studies using BMMNCs from patients with MDS and AML to examine the effects of the styryl sulfone ON 01910.Na on cyclin D1 accumulation, aneuploidy, and CD34+ blast percentage. We next treated twelve patients with higher risk MDS and two trisomy 8 AML patients with ON 01910.Na on a phase I clinical protocol (NCT00533416).. ON 01910.Na inhibited cyclin D1 expression, and was selectively toxic to trisomy 8 cells in vitro. Flow cytometry studies demonstrated increased mature CD15+ myeloid cells and decreased CD34+ blasts. Three patients treated with ON 01910.Na on a clinical had decreased bone marrow blasts by ≥ 50%, and three patients had hematologic improvements, one of which was sustained for 33 months. Patients with hematologic responses to ON 01910.Na had decreased cyclin D1 expression in their CD34+ cells.. The preclinical results and responses of patients on a clinical trial warrant further investigation of ON 01910.Na as a potential novel targeted therapy for higher risk MDS patients.

Topics: Aged; Aged, 80 and over; Algorithms; Antineoplastic Agents; Bone Marrow Cells; Chromosomes, Human, Pair 8; Cyclin D1; Dose-Response Relationship, Drug; Female; Glycine; Humans; Male; Middle Aged; Molecular Targeted Therapy; Myelodysplastic Syndromes; Sulfones; Trisomy; Tumor Cells, Cultured

Myelodysplastic syndromes (MDS) are characterized by ineffective hematopoiesis and may progress to acute myeloid leukemia (AML). MicroRNAs (miRNA/miRs) as oncogenes or tumor suppressors regulate a number of biological processes including cell proliferation, cell cycle and apoptosis in different types of cancer cells. Recently, it has been reported that miR‑21 as an oncogene is overexpressed and directly targets SMAD‑7 in MDS. However, little is known about the mechanism of miR‑21 in the progression of MDS. In the present study, the role of miR‑21 in the proliferation and apoptosis of SKM‑1 cells, an acute myeloid leukemia cell line established in the AML/MDS leukemic phase was investigated. The present results demonstrated that downregulation of miR‑21 inhibited proliferation, induced apoptosis and caused G1 phase cell cycle arrest of SKM‑1 cells. In addition, the expression levels of apoptosis regulator Bcl‑2 (bcl2), cyclinD1 and phosphorylated‑protein kinase B (AKT) were significantly decreased in SKM‑1 cells transfected with the miR‑21 inhibitor, whilst the expression levels of phosphatase and tensin homolog (PTEN), bcl‑associated protein X (bax) and cleaved caspase 3 were significantly elevated. Furthermore, knockdown of Akt by small interfering (si)RNA significantly increased the expression of bax, cleaved caspase 3 and reduced the expression of bcl2 and cyclinD1 in SKM‑1 cells. Taken together, these data indicate that miR‑21 targets the PTEN/AKT pathway in the pathogenesis of MDS and could be a potential target for MDS therapy.

Topics: Antagomirs; Apoptosis; bcl-2-Associated X Protein; Caspase 3; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Down-Regulation; G1 Phase Cell Cycle Checkpoints; Humans; MicroRNAs; Myelodysplastic Syndromes; Proto-Oncogene Proteins c-akt; Proto-Oncogene Proteins c-bcl-2; PTEN Phosphohydrolase; RNA Interference; RNA, Small Interfering; Signal Transduction

Myelodysplastic syndromes (MDS) are a group of heterogeneous diseases characterized by poorly formed blood cells. We wanted to elucidate the underlying molecular mechanism to better determine pathogenesis, prognosis, diagnosis, and treatment for patients with MDS. We compared gene expression levels between normal and MDS tissue samples by immunohistochemical analysis. We studied the proliferation, survival, and migration of MDS cells using the EDU assay, colony formation, and transwell assays. We assessed the apoptotic rate and cell cycle status using flow cytometry and Hoechst staining. Finally, we evaluated RNA and protein expressions using polymerase chain reaction and Western blots, respectively. We found that resveratrol suppressed SKM-1 (an advanced MDS cell line) proliferation in a dose-dependent manner. Consistent with this finding, the EDU and colony formation assays also showed that resveratrol inhibited SKM-1 growth. Moreover, flow cytometry and Hoechst 33258 staining demonstrated that resveratrol induced apoptosis and a change in cell cycle status in SKM-1 cells, while the transwell assay showed that resveratrol reduced the migratory ability of SKM-1 cells. Resveratrol also decreased the expression of CCND1 (a gene that encodes the cyclin D1 protein) and increased expressions of KMT2A [lysine (K)-specific methyltransferase 2A] and caspase-3, suggesting that resveratrol exerts its effect by regulating CCND1 in SKM-1 cells. In addition, a combination of resveratrol and the PI3K/AKT inhibitor LY294002 exhibited a stronger inhibitory effect on the SKM-1 cells, compared with resveratrol alone. Our study proved that resveratrol suppresses SKM-1 growth and migration by inhibiting CCND1 expression. This finding provides novel insights into the pathogenesis of MDS and might help develop new diagnosis and treatment for patients with MDS.

Topics: Antineoplastic Agents, Phytogenic; Apoptosis; Case-Control Studies; Cell Cycle; Cell Movement; Cell Proliferation; Cyclin D1; Gene Expression Regulation; Humans; Myelodysplastic Syndromes; Resveratrol; Stilbenes; Tumor Cells, Cultured

Topics: Cyclin D1; Female; Glycine; Humans; Male; Molecular Targeted Therapy; Myelodysplastic Syndromes; Sulfones

The aim of this study was to investigate the inhibition effect of arsenic sulfide (As2S2) on the growth of in vitro cultured BMMNC from MDS patients and to explore its possible cellular and molecular mechanisms. The apoptosis of MDS cells induced by As2S2 solution of different concentrations were studied with MTT, flow cytometry, and RT-PCR. The results showed that (1) low concentration of As2S2 (0-0.6 mg/L) had no marked inhibition effect on proliferation of MDS cells; (2) after treatment with 1.5-50 mg/L of As2S2, both low risk MDS cells and high risk MDS cells presented typical features of apoptosis with a dose-dependent manner, the expression of bcl-2 mRNA and the ratio of bcl-2/bax obviously decreased after As2S2 treatment (P < 0.05); (3) BMMNC from MDS patients had higher apoptosis ratio than that of BMMNC from control. It is concluded that BMMNC excessive apoptosis exists in MDS patients; low concentration of As2S2 (0-0.6 mg/L) shows no inhibition effect on proliferation of MDS cells; high concentration of As2S2 (1.5-50 mg/L) induces apoptosis of MDS cells.

Topics: Adult; Aged; Aged, 80 and over; Antineoplastic Agents; Apoptosis; Arsenicals; bcl-2-Associated X Protein; Bone Marrow Cells; Cyclin D1; Female; Humans; Leukocytes, Mononuclear; Male; Middle Aged; Myelodysplastic Syndromes; RNA, Messenger; Sulfides

An unusually high incidence of apoptosis in S-phase cells is characteristically found in the bone marrow (BM) of patients with myelodysplastic syndromes (MDS). Previously, E2F1, c-myc, and Cyclin D1 have been shown to bring about both S-phase changes and/or apoptotic changes. We have already found a stoichiometric imbalance between pRb and E2F1 causing deregulated E2F1 activity in these disorders. In the present study, we investigated the status of Cyclin D1 in relation to E2F1 and apoptosis in 19 patients with a confirmed diagnosis of MDS in comparison with 6 healthy donors. Cyclin D1 was localized immunohistochemically using a specific monoclonal antibody (1:150 dilution) in plastic-embedded BM sections. The nuclear localization of Cyclin D1 graded on a subjective rating scale of 0 (negligible staining) to 8+ (highest), demonstrated negligible levels in normal marrows (median 1+), and in 11/19 evaluable MDS marrows. In contrast, 8/19 MDS biopsies showed an almost four-fold increase in Cyclin D1 localization (p< or =0.001). A western blot analysis of E2F1 in corresponding bone marrow (BM) aspirate mononuclear cells (MNC) demonstrated that the MDS patients with elevated Cyclin D1 expression also had a significant increase in E2F1 protein (p< or =0.03). Additionally, these patients revealed higher levels of mRNA of one of the E2F1 transcriptional target genes, dihydrofolate reductase (DHFR, p=0.01). Subsequently, the relationship of Cyclin D1 with apoptosis was elucidated in a colocalization experiment in BM biopsy sections using immunohistochemistry for Cyclin D1 and in situ end labeling of DNA (ISEL) for apoptosis. The percentage of ISEL-positive apoptotic cells was several fold higher in MDS as compared to normal BMs (p=0.009). Interestingly, 7-41% (median 20%) of the apoptotic cells in different MDS BMs revealed co-localization of Cyclin D1 in their nucleus, whereas in normal BMs co-localization was virtually absent (p=0.008). Thus, it is possible that in a subset of MDS patients, apoptotic death of bone marrow cells may involve Cyclin D1/E2F1 pathway.

Topics: Adult; Aged; Antibodies, Monoclonal; Apoptosis; Blotting, Western; Bone Marrow Cells; Cell Cycle Proteins; Cell Nucleus; Cyclin D1; DNA-Binding Proteins; E2F Transcription Factors; E2F1 Transcription Factor; Female; Humans; Immunohistochemistry; Male; Middle Aged; Myelodysplastic Syndromes; Retinoblastoma Protein; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; S Phase; Transcription Factors; Transcription, Genetic

The role of positive and negative cytokine interactions in G1 cell cycle regulation of haemopoietic cells was analysed by determination of the expression patterns of D-type cyclins and cyclin-dependent kinases (cdks) in SKM-1 myelodysplastic syndrome (MDS) cells incubated with granulocyte-macrophage colony-stimulating factor (GM-CSF) and/or transforming growth factor-beta 1 (TGF-beta 1). TGF-beta 1 inhibited SKM-1 cell proliferation due to the cell cycle arrest in G1 phase. GM-CSF abrogated the TGF-beta 1-mediated G1 arrest in these cells. Reverse transcription-polymerase chain reaction (RT-PCR) analysis indicated that TGF-beta 1-mediated G1 arrest correlated with the down-regulation of cdk4, cdk6 and cyclin D2, and that abrogation of TGF-beta 1-mediated G1 arrest by GM-CSF correlated with the constitutive over-expression of cyclin D2 and cdk6 but not cdk4. These results suggest the importance of cyclin D2/cdk6 levels in abrogating G1 arrest in cells exposed to TGF-beta 1, and raise the possibility that the GM-CSF-mediated up-regulatory pathway of signal transduction through cyclin D2/cdk6 differs from the TGF-beta 1-cdk4-mediated pathway in SKM-1 cells. This signal transduction pathway through cyclin D2/cdk6 might play an important role in haemopoietic regulation by the cytokine network.

Topics: Cell Cycle; Cell Division; Cyclin D1; Cyclin D2; Cyclin D3; Cyclin-Dependent Kinase 6; Cyclin-Dependent Kinases; Cyclins; Drug Interactions; Granulocyte-Macrophage Colony-Stimulating Factor; Humans; Myelodysplastic Syndromes; Protein Serine-Threonine Kinases; Transforming Growth Factor beta; Tumor Cells, Cultured; Up-Regulation