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[8-Chloro-adenosine increases the activity of transcription factor CREB by modification of phosphorylation]
Xiao-Yan Chang1, Jia-Chun Fang, Nai-Qin Wang
1Department of Cell Biology, Beijing Institute for Cancer Research,School of Oncology, Peking University, Beijing, PRChina. changxiaoyan2002@yahoo.com.cn
Background & Objective:
8-Chloro-adenosine(8-Cl-Ado) is an anti-tumor drug,which has been investigated and developed recently in China. The molecular mechanism of 8-Cl-Ado is now unclear. This study was conducted to investigate the effect of 8-Cl-Ado on the activity of transcription factor cAMP-response-element-binding protein(CREB) and its correlative mechanism in human promyelocytic leukemia cell line HL-60.
Methods:
MTT method was used to determine IC(50) (the concentration showing 50% of inhibition rate) of 8-Cl-Ado to HL-60. The protein levels and phosphorylated levels of CREB at different time following 8-Cl-Ado stimulation were examined using Western blot analysis. Gel shift assay was performed to examine DNA binding levels of CREB at different time following 8-Cl-Ado stimulation.
Results:
(1)The IC(50) of 8-Cl-Ado to HL-60 was about 0.42 micromol/L. (2)8-Cl-Ado increased phosphorylated level of CREB rapidly, which was maximal at 30 minutes, and returned to basal level in 6 hours. The protein level of CREB was constant during 8-Cl-Ado stimulation. (3)8-Cl-Ado did not influence binding level of DNA and CREB.
Conclusion:
The increase of transcriptional activity of CREB induced by 8-Cl-Ado depends on the increased level of phosphorylated CREB, but is not associated with the protein level and DNA binging level of CREB.