[SHP-1 gene's methylation status of Daudi lymphoma cell and the demethylation effect of 5-aza-2'-deoxycytidine].
OBJECTIVE
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.


METHODS
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.


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.


CONCLUSIONS
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.
Fang
Jian-chen
JC
Department of Pathology the Third Hospital, Sun Yat-sen University, Guangzhou 510630, China.
Su
Zu-lan
ZL
Qiu
Geng
G
He
Dan
D
Feng
Zhi-ying
ZY
Zhu
Ming-fen
MF
Li
Li
L
He
Xiang-lei
XL
D000964:Antimetabolites, Antineoplastic / Q000494:pharmacology*; D017209:Apoptosis; D001374:Azacitidine / Q000031:analogs & derivatives* / Q000494:pharmacology; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects*; D019175:DNA Methylation; D000077209:Decitabine; D004305:Dose-Response Relationship, Drug; D006801:Humans; D008223:Lymphoma / Q000235:genetics / Q000473:pathology; D053824:Protein Tyrosine Phosphatase, Non-Receptor Type 6 / Q000096:biosynthesis / Q000235:genetics*; D012333:RNA, Messenger / Q000096:biosynthesis; D020133:Reverse Transcriptase Polymerase Chain Reaction
D000964:Antimetabolites, Antineoplastic; D012333:RNA, Messenger; D000077209:Decitabine; D053824:Protein Tyrosine Phosphatase, Non-Receptor Type 6; D001374:Azacitidine