| OBJECTIVEThe myelodysplastic syndromes (MDS) are a group of disorders characterized by peripheral pancytopenia despite normo- or hyper-cellular bone marrow. Morphological evidence of increased apoptosis in marrow hemopoietic cells has been demonstrated in patients with MDS. Early in MDS, increased apoptosis is associated with ineffective progenitor and maturing hemopoietic cell survival, and occurs concomitant with cytopenias/ineffective hemopoiesis; conversely, the progression of MDS toward AML occurs in concert with decreased apoptosis and an increased degree of neoplastic cell survival, leading to subsequent expansion of the abnormal precursor cells. Clinical heterogeneity has been demonstrated in MDS, with morphologically 'early' MDS patients having 5% marrow blasts [refractory anemia (RA) or RA with ringed sideroblasts (RARS)], having better prognosis and a lower potential for evolution to AML than those 'late' MDS patients with 5-30% of marrow blasts (refractory anemia with excess (RAEB) or RAEB in transformation (RAEB-T)). Despite overwhelming evidence for a role of aberrant apoptosis in myelodysplasia, the molecular mechanisms responsible for such changes have not been elucidated. Apoptosis (programmed cell death) is an active cellular processwhich regulates cell population size by decreasing cell survival. These processes are associated with alterations in the balance between pro- and anti-apoptotic oncoprotein expression within the hemopoietic precursors. The inhibitor of apoptosis (IAP) gene family, which was discovered less than a decade ago, encodes a group of structurally related proteins that, in addition to their ability to suppress apoptotic cell death, are involved in an increasing number of seemingly unrelated cellular functions. The IAP families have evolved as highly conserved regulators of cell death.. Homoharringtonine(HHT) is a plant alkaloid with antileukemia activity which is currently tested for treatment of acute and chronic leukemia. Our previous study suggested that HHT could induced apoptosis of MUTZ-1 in vitro, but little is know about the possible molecular mechanisms of HHT induced MUTZ-1 cell apoptosis. The study on the mechanisms of HHT in regulation of apoptosis in MUTZ-1 cells will give us insight into further understanding of the role HHT. The data generated from this study will also provide theoretical ground for MDS were treated with HHT.MATERIALS AND METHODS1 In this study, five cell lines including MUTZ-1, K562, Jurkat, RMPI and HL60 were used in the experiments to detect the effects of HHT on the induction of apoptosis and to study the possible mechanisms of HHT in regulation of apoptosis of the cells. Cell apoptosis and cell cycle shift were observed by flow cytometry (FCM). Cell apoptotic morphology was observed by transmission electron microscope.2 Semi-quantitative RT-PCR was used to evaluate the mRNA expression of survivin, XIAP, Bcl-2 and Bax in the MDS patients, and cell lines treated with HHT.3 As-ODN transfection was used to analyse the effects of survivin As-ODN in MUTZ-1 cell treated with HHT.RESULTS1 The results demonstrated that HHT was capable of inducing apoptosis of MUTZ-1, K562, Jurkat, RMPI and HL60 cell lines, but the apoptotic rate of different cell lines were different when these cell lines were treated with same concertration HHT. The apoptotic rate of K562 was lower than that of MUTZ-1 and RMPI (P<0.05); the apoptotic rate of MUTZ-1 and RMPI were lower than that of Jurkat and HL60 (P<0.05); there was no significance between MUTZ-1 and RMPI or between Jurkat and HL60. We examined the exposition of phosphatidylserine (PS) on the cell surface by using annexin V-FITC and PI. After 6, 12, and 24h culture with HHT (0.05, 0.2, 0.4, 0.8g/ml), the cell apoptosis was shown in a dose- and time- dependent manner. The survivin mRNA was detected in five cell lines. K562 had a higher survivin mRNA expression than MUTZ-1 and RMPI (P<0.05), MUTZ-1 and RMPI had a higher survivin mRNA expression than Jurkat and HL60... |
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