| Background and ObjectionIron is an essential factor in many important cell functions, including growth, immunological response and energy production. It is important to maintain cellular iron homeostasis for cell proliferation and normal function. The iron regulatory proteins (IRP1 and IRP1) and the ferritin (Fn) and the transferrin receptor (TfR) involved in the maintenance of cellular iron homeostasis. Fn and TfR are used by cells to adjust intracellular iron concentration to levels in order to be adequate for their metabolic needs. IRP1 and IRP2 might bind to transcripts of ferritin, transferrin receptor to control the expression of iron metabolism proteins at the post-transcriptional level.Tumor cells have a close relation with iron, as other cells do. And leukemia is one of common malignancies. The studies showed iron play a role in the development of leukemia. Although post-transcriptional gene regulation by the interaction of IRPs and IRE on select mRNAs is a very active research area, there is relatively little data on gene transcription which may be modified by IRP2 underdifferent iron status. In order to reveal the mechanism of iron metabolism and the roles of iron playing in leukemia ,we investigated the expression levels and the relationship of IRP2mRNA, TfRmRNA , Fn mRNA and WT1 mRNA under different iron status of HL-60 cells, and probed into the roles of IRP2 playing in HL-60 cells.MethodsHL-60 cells (in 24-well plates) were cultured in RPMI 1640 medium supplemented with 10% heat-inactivated fetal bovine serum at 37 C in 5% CO2-95% air with 100 U/ml penicillin G and 100 U/ml streptomycin. To examine effects of iron, HL-60 cells were divided into five groups which treated with ferric chloride(FeCL3) or deferoxamine(DFO). HL-60 cells in the control group cultured in control media, in FeCL3-20 group cultured in control media plus of 20 M/L FeCL3, in FeCL3-40 group cultured in control media plus of 40 M/L FeCL3, in DFO-50 group cultured in control media plus of 50 M DFO and in DFO-100 group cultured in control media plus of 100 M DFO. The cells were harvested at 12, 24 and 48h proliferation, and total RNA was isolated; cDNA was synthesized by reverse transcription (RT), and relative amounts of IRP2 mRNA , Fn mRNA and TfR mRNA were determined by RT-PCR.Results1. The levels of IRP2mRNA remained constant in all cells, whether or not treated with DFO or FeCL3.However, they were decreased when the cells prolonged in the media. There was not significant difference among the between-subjects (F=1.199,P > 0.05),but there was significant difference among the within-subjects (F=43.418, P<0.01).2. The levels of TfRmRNA increased in the cells treated with DFO and they are dose-time dependant. In contrast to the control cells which treated neither with DFO nor ferric chloride ,there was significant difference (Fw-s = 7.184, FB-S =113.926; P<0.01). Surprisingly, when cells grown with FeCLs, there wasn't a decline in expression of TfR mRNA, but it increased lightly at 12 hours andpeaked at 24 hours. This was followed by a drastically decline in its expression so that by 48 hours the levels of TfR mRNA were approximately two times fewer in the cells grown in the presence of 40 uM/L FeCLs than in the control cells. There was significant difference (P <0.01).3. There was not significant difference among the within-subjects (F= 1.514.P > 0.05). It suggested that the levels of Fn mRNA didn't dependent on time.However, there was significant difference among the between-subjects (F=209.056, P<0.01).The levels of Fn mRNA in the cells treated with FeCLswere approximately two times in the control cells. In contrast with the control cells, there was significant difference (P <0.05). The levels of Fn mRNA of the cells grown in DFO had little change. In contrast to the control cells, there was not significant difference (P > 0.05).4. There wasn't significant correlation between IR?2 mRNA and TfR mRNA,Fn mRNA in HL-60 cells (P > 0.05 ).5. The levels of WT| mRNA remained constant in all cells whether or not... |
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