SHP-1 gene's methylation status of Daudi lymphoma cell and the demethylation effect of 5-aza-2'-deoxycytidine.
- Author:
Jian-chen FANG
1
;
Zu-lan SU
;
Geng QIU
;
Dan HE
;
Zhi-ying FENG
;
Ming-fen ZHU
;
Li LI
;
Xiang-lei HE
Author Information
- Publication Type:Journal Article
- MeSH: Antimetabolites, Antineoplastic; pharmacology; Apoptosis; Azacitidine; analogs & derivatives; pharmacology; Cell Line, Tumor; Cell Proliferation; drug effects; DNA Methylation; Dose-Response Relationship, Drug; Humans; Lymphoma; genetics; pathology; Protein Tyrosine Phosphatase, Non-Receptor Type 6; biosynthesis; genetics; RNA, Messenger; biosynthesis; Reverse Transcriptase Polymerase Chain Reaction
- From: Chinese Journal of Hematology 2006;27(10):670-674
- CountryChina
- Language:Chinese
-
Abstract:
OBJECTIVETo explore the transcription regulation of 5-aza-2'-deoxycytidine(5-Aza-CdR) on SHP-1 gene and its effects on Daudi cell line growth.
METHODSMTT 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.
RESULTS(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 dependent:after 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, too:at 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.
CONCLUSIONDNA 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.