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[Growth and gene expression of leukemia cell after treated with methylation inhibitor 5-aza-2'-deoxycytidine]
Shu-kai Qiao1, Shi-rong Xu, Xiao-nan Guo
1The Second Hospital, Hebei Medical University, Shijiazhuang 050000, China.
Objective:
To investigate the mechanism of demethylation therapy of leukemia by 5-aza-2'-deoxycytidine (5-aza-CdR).
Methods:
By using MTT test, NBT reduction reaction and DNA agarose gel electrophoresis, changes in proliferation, differentiation and apoptosis were observed in K562, HL-60 and fresh leukemia cells after treated with 5-aza-CdR. The mRNA expressions of DNMTs, p15, p53 and bcl-2 were measured by RT-PCR. The status of p15(INK4B) gene methylation was examined by methylation-specific PCR (MSP-PCR).
Results:
The growth inhibition of K562, HL-60 and fresh leukemia cells displayed a dose and time-dependent manner after treated by 5-aza-CdR. The differentiation-inducing ability on HL-60 cells was obvious at 0.5 micromol/L of 5-aza-CdR. The up-regulation of p15 mRNA and p53 mRNA expression and down-regulation of bcl-2 mRNA expression were obvious as compared with the control, but the DNMTs expression was not significantly different from the control. The methylation status of p15 gene in fresh leukemia cells decreased gradually with increasing concentration of 5-aza-CdR.
Conclusion:
The proliferation of leukemia cells was obviously inhibited by 5-aza-CdR, its mechanism maybe related to the up-regulation of p15 and p53 genes and down-regulation of bcl-2 gene. The decrease of p15 gene methylation was associated with the competitive inhibition of 5-aza-CdR.
Insights
5-aza-2'-deoxycytidine (5-aza-CdR) inhibits leukemia cell proliferation by upregulating p15 and p53 genes and downregulating bcl-2. This demethylation therapy reduces p15 gene methylation, offering a potential treatment strategy for leukemia.
Area of Science:
- Oncology
- Molecular Biology
- Pharmacology
Background:
- Leukemia treatment often involves targeting cellular proliferation and differentiation.
- Demethylation therapy, using agents like 5-aza-2 -deoxycytidine (5-aza-CdR), is explored for its potential in leukemia treatment.
- Understanding the precise mechanisms of 5-aza-CdR is crucial for optimizing its therapeutic application.
Purpose of the Study:
- To elucidate the mechanism by which 5-aza-CdR exerts its demethylation effects in leukemia.
- To investigate the impact of 5-aza-CdR on leukemia cell proliferation, differentiation, and apoptosis.
- To analyze the effects of 5-aza-CdR on key gene expressions and methylation status in leukemia cells.
Main Methods:
- Cell viability and proliferation were assessed using MTT assays.
- Cell differentiation was evaluated through NBT reduction assays.
- Apoptosis and DNA changes were observed using DNA agarose gel electrophoresis.
- mRNA expression levels of DNMTs, p15, p53, and bcl-2 were quantified via RT-PCR.
- Methylation-specific PCR (MSP-PCR) was employed to examine the methylation status of the p15(INK4B) gene.
Main Results:
- 5-aza-CdR demonstrated dose- and time-dependent growth inhibition in K562, HL-60, and fresh leukemia cells.
- Significant differentiation induction was observed in HL-60 cells at 0.5 micromol/L of 5-aza-CdR.
- Up-regulation of p15 and p53 mRNA and down-regulation of bcl-2 mRNA were noted, while DNMTs expression remained unchanged.
- A gradual decrease in p15 gene methylation was observed with increasing 5-aza-CdR concentrations in leukemia cells.
Conclusions:
- 5-aza-CdR effectively inhibits leukemia cell proliferation, likely through the upregulation of p15 and p53 genes and downregulation of the bcl-2 gene.
- The observed decrease in p15 gene methylation is associated with the mechanism of 5-aza-CdR, suggesting competitive inhibition.
- These findings highlight the potential of 5-aza-CdR as a demethylation therapeutic agent for leukemia.
