SHP-1 gene's methylation status of Daudi lymphoma cell and the demethylation effect of 5-aza-2'-deoxycytidine / 中华血液学杂志

ABSTRACT

<p><b>OBJECTIVE</b>To explore the transcription regulation of 5-aza-2'-deoxycytidine(5-Aza-CdR) on SHP-1 gene and its effects on Daudi cell line growth.</p><p><b>METHODS</b>MTT method and flow cytometry were used to detect the growth and apoptosis of Daudi cells after treated with different dosage of 5-Aza-CdIR. Bisulfite sequencing PCR ( BSP) , T-A cloning and sequence analysis were evaluated for methylation status. The SHP-I mRNA and protein were determined by reverse transcription polymerase chain reaction (RT-PCR) ,immunohistochemistry.</p><p><b>RESULTS</b>(1)After 7 d treatment with 2. 00 micromol/L of 5-Aza-CdR, all cytosines (C) in Daudi cells genome DNA were converted to thymidine, and SHP-1 mRNA and protein expressed again in the cells while those Cs in CpG dinucleotides in untreated Daudi cells remained Cs; (2)5-Aza-CdR inhibited the cell growth,The effects within certain extent were dose and time dependentafter 72 h treatment with 5-Aza-CdR at 200. 00, 20. 00, 2. 00 and 0. 20 micromol/L, the inhibitive rates were 72. 0% , 65. 1%, 51. 5%, 28.8% ,23.4% respectively; (3) 5-Aza-CdR increased apoptosis rate of tumor cells with a dose and times dependent manner within certain extent, tooat the 1,3,5 d treatment with 5-Aza-CdR 2. 00 micromol/L,the apoptosis rates were 2. 3% ,10. 8 % and 17. 1% ; respectively. (4) 5-Aza-CdR also changed cell cycle of tumor cells at 24 h treatment with 5-Aza-CdR 2.00 micromol/L,92. 7% tumor cells stopped at S phase and G, phase cells were increased gradually with time.</p><p><b>CONCLUSION</b>DNA promoter hypermethylation is associated with SHP-1 gene silence in Daudi lymphoma cell line. 5-Aza-CdR could effectively cause demethylation and inhibit the growth of tumor cell by reactivating the gene transcription.</p>