| Part1The impact of DNA methyl trans f erase inhibitor Decitabine on P15INK4B gene methylation status in SKM-1cellsObjective:To explore the demethylation effect of P15INK4Bgenein myelodysplastic syndrome(MDS) transformation to leukemia cell line SKM-linduced by DNA methyltransferase inhibitorDecitabine(DAC).Methods:SKM-1cells were treated with1.5,3. Oand6. Oμmol/L DACfor48h, untreated SKM-1cellsas the control group. The methylation of P15INK4B gene, the expression of P15INK4B gene mRNA, the cellular proliferation, the cell cycleand the cellular early apoptosiswere detected by methylation specific PCR(MSP),realtime RT-PCT, CFSE methods, propidium iodide(PI) staining and annexin V-FITC/PI double labeling.Results:(1)The control group wastotally methylated with P15INK4B gene, without any unmethylatedstrip. While1.5μmol/L,3.0μmol/Land6.0μmol/L DAC treated groupswere partial methylation, and showing unmethylatedstrip. The degree of P15INK4B gene methylation was lower in the groups with the higher concentrations of DAC.(2) The expression of P15INK4B gene mRNA in the control group,1.5μmol/L,3.0μmol/L and6.0μmol/L groups werel.00±0.12,0.91±0.13,0.51±0.06and0.43±0.04, respectively. Compared with control group, there were statistical significances in3.Oμmol/L and6. Oumol/L groups(P<0.05). With the paired comparison inDAC groups, there were statistical significances between3.0μmol/Land1.5μmol/Lgroups,6.0μmol/Land1.5μmol/Lgroups (P<0.05), butthere was no statistical significance between6. Oμmol/L and3.0μmol/Lgroups(P>0.05).(3) The MFI of CFSE in the control group,1.5μmol/L,3.0μmol/L and6.0μmol/L groups were51.67±1.61,55.33±2.28,56.33±2.50and55.71±2.87, respectively. Compared with control group, the3.0μmol/L group was significantly decreased(P<0.05), there wereno statistical significances inl.5μmol/L and6.0μmol/L group(P>0.05). Also no statistical significances with the paired comparison inDAC groups(P>0.05).(4)The proportionofGl phase cells in the control group,1.5μmol/L,3. Oμmol/L and6.0μmol/L groups were55.78±3.61%,48.12±2.93%,51.69±1.60%and46.71±1.54%, respectively. The proportionofS phase cellsin the control group,1.5μmol/L,3.0μmol/L and6.0μmol/L groupswere44.22±3.61%,51.88±2.93%,48.31±1.60%and53.29±1.54%, respectively. Compared with control group, the proportionofGl phase cells in1.5μmol/L group and6.0μmol/L group were decreased significantly (P<0.05). With the paired comparison in DAC groups, there was statistical significance between6.0μmol/L group and3.0μmol/L groups(P<0.05).(5)The early apoptosis ratesin the control group,1.5μmol/L,3.0μmol/L and6. O)μmol/L group were2.91±0.26%,7.77±0.22%,12.45±0.28%and13.86±0.27%, respectively. Compared with control group, the early apoptosis ratesin DAC groups were increased significantly (P<0.05). There were statistical significance with the paired comparison inDAC groups (P<0.05).Conclusion:DAC could induce the demethylation of P15INK4B gene, suppress cellular proliferation, arrest cells in S phase and increased cellular early apoptosis in SKM-1cells. But we didn’t found P15INK4B gene mRNA re-expression withP15INK4B genedemethylation. Part2The impact of Defetoxamineon P15INK4B gene methylation status in SKM-1cellsObjective:To explore the demethylation effect of P15INK4B genein SKM-lcells induced by iron chelating agentsDeferoxamine(DFO).Methods:SKM-1cells were treated withferric ammonium citrate(FAC) and DFO, divided into three groups:control group, FAC group and FAC+DFO group. Cellular labile iron pool(LIP), reactive oxygen species(ROS), the methylation of P15INK4B gene, the expression of P15INK4B gene mRNA, the cellular proliferation, the cell cycleand the cellular early apoptosis were detected by Calcein-AM staining, ROS detection kit, MSP, realtime RT-PCT, CFSE methods, PIstaining and annexin V-FITC/PI double labeling.Results:P15INK4B gene in control group and FAC group were totally methylated, without any unmethylatedstrip. While FAC+DFO group was partial methylation, showing unmethylatedstrip. Compared with control group, the LIP(64.04±2.12vs1.45±0.65)%and ROS(45.57±1.18vs33.38±12.96)%were increased significantly, MFI of CFSE (23.01±5.20vs51.67±1.61)%, P15INK4Bgene mRNA level (0.72±0.08vs1) and early apoptosis rate(1.76±0.11vs2.91±0.25)%were decreased significantly in FAC group (P<0.05). Compared with FAC group, the LIP(8.34±4.21vs64.04±2.12)%and ROS(34.39±2.12vs45.57±1.18)%were decreased significantly, MFI of CFSE(37.34±6.61vs23.01±5.20)%, P15INK4B gene mRNA level (1.50±0.15vs0.72±0.08) and early apoptosis rate(4.77±0.88vs1.76±0.11)%were increased significantly in FAC+DFO group (P<0.05). The differences of cell cycleamong3groups were no statistical significances.Conclusions:Iron chelator agent DFO could decrease LIP and ROS, induce the demethylation of p15INK4B gene, re-expressionp15INK4B gene mRNA, suppress cellular proliferation, and increase cellular apoptosis in iron-overloaded SKM-1cells. Part3The impact of Decitabine combined with Defetoxamineon P15INK4B gene methylation status in SKM-1cellsObjective:To explore the demethylation effect of P15INK4Bgenein SKM-lcells induced by DAC combined with DFO.Methods:SKM-1cells were treated with FAC, DFO, DAC, and divided into four groups:control group, FAC group, FAC+DFO group and FAC+DFO+DAC group. ROS, the methylation of P15INK4B gene, the expression of P15INK4B gene mRNA, the cellular proliferation, the cell cycleand the cellular early apoptosis were detected. Results:(1) The MFI of ROS in the control group, FAC, FAC+DFO and FAC+DFO+DAC groups were33.38±12.96,45.57±1.18,34.39±2.12and36.64±1.96, respectively. Compared with FAC group, the FAC+DFO group and FAC+DFO+DAC group were significantly decreased(P<0.05);(2) In MSP, the control group and FAC group weretotally methylated with P15INK4B gene, without any unmethylatedstrip. While FAC+DFO group and FAC+DFO+DAC group were partial methylation, and showing unmethylatedstrip. The degree of P15INK4B gene methylation was reversed in some extent.(3) The expression of P15INK4B gene mRNA in the control group, FAC, FAC+DFO and FAC+DFO+DAC groups were1.00±0.12,0.71±0.08,1.49±0.15andl.31±0.13, respectively. Compared with FAC group, the FAC+DFO group and FAC+DFO+DAC group were significantly increased (P<0.05);(4) The MFI of CFSE in the control group, FAC, FAC+DFO and FAC+DFO+DAC groups were51.67±1.61,23.01±5.20,37.34±6.61and39.61±6.92, respectively. Compared with FAC group, the FAC+DFO group and FAC+DFO+DAC group were significantly increased(P<0.05);(5) The proportionofGl phase cells in the control group, FAC, FAC+DFO and FAC+DFO+DAC groups were55.78±3.61%,50.2±9.20%,51.10±6.30%and49.58±3.38%, respectively. The proportionofS phase eellsin the the control group, FAC, FAC+DFO and FAC+DFO+DAC groups were44.22±3.61%,49.77±9.20%,48.90±6.30%and50.42±3.38%, respectively. Compared with FAC group, the proportionofGl phase cells in the FAC+DFO group and FAC+DFO+DAC groups were decreased, and the proportionofS phase cells in them were increased. But the differences of cell cycle among4groups were no statistical signif icances(P>0.05).(6)The early apoptosis rates in the control group, FAC, FAC+DFO and FAC+DFO+DAC groups were2.91±0.26%,1.76±0.11%,4.77±0.88%and8.94±0.87%, respectively. Compared with the FAC and FAC+DFO groups, the early apoptosis rate in FAC+DFO+DAC group was increased significantly(P<0.05). And the result of TUNEL stainingobserved by fluorescence microscopy also proved that the early apoptosis cells were increasedin FAC+DFO+DAC group.Conclusion:The FAC+DAC+DFOgroup shows no difference with FAC+DFO group in ROS、the demethylation of P15INK4B gene、the expression of P15INK4B gene diRNA、 cellular proliferation and cell cycle, but increased of early cellular apoptosis in iron-overloaded SKM-1cells. |
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