[5-azacytidine treatment induces tumor suppressor gene XAF1 expression and inhibits proliferation in myeloma cells].
OBJECTIVE
To investigate the effect of 5-azacytidine on XAF1 expression in myeloma cell lines RPMI8226 and XG-7 and the in vitro anti-myeloma activity of 5-azacytidine.


METHODS
XAF1 mRNA and protein expression was detected by semi-quantitative reverse transcriptase PCR and Western blot, respectively. Methylation specific PCR (MSP) was used to detect methylation status of XAF1 promoter CpG islands. RPMI8226 and XG-7 cells were treated with 0-5 micromol/L of 5-azacytidine and Cell Counting Kit-8 colorimetric assay was used to evaluate the growth inhibitory effect. Cell apoptosis was determined with Annexin V-PE/7-AAD staining by flow cytometry.


RESULTS
Untreated RPMI8226 cells expressed XAF1 mRNA isoforms 1 and 2, and untreated XG-7 cells had no XAF1 expression. Hypermethylation of XAF1 promoter CpG islands was detected in both the cell lines. After treated with 2.5 micromol/L 5-azacytidine for 72 h, both the cell lines expressed full-length XAF1 transcript and protein. 5-azacytidine treatment led to XAF1 promoter CpG islands hypomethylation and showed anti-myeloma activity in a time- and concentration-dependent manner with IC50 of 2.4 micromol/L and 2.6 micromol/L at 48 h for RPMI8226 and XG-7 cell lines, respectively.


CONCLUSIONS
Lack of XAF1 expression and abnormal expression of XAF1 in myeloma cell lines are associated with XAF1 gene promoter CpG islands hypermethylation. 5-azacytidine treatment can induce XAF1 mRNA and protein expression and exerts anti-myeloma activity via apoptosis at clinically achievable concentrations.
Chen
Guang-Hua
GH
Jiangsu Institute of Hematology, Key Laboratory of Thrombosis and Hemostasis, Ministry of Health, First Hospital of Soochow University, Suzhou 215006, China.
Lin
Feng-Ru
FR
Wu
De-Pei
DP
Wang
Yi
Y
Huang
Hai-Wen
HW
Tang
Xiao-Wen
XW
Zhu
Zi-Ling
ZL
Feng
Yu-Feng
YF
Chang
Wei-Rong
WR
D048868:Adaptor Proteins, Signal Transducing; D017209:Apoptosis / Q000187:drug effects; D051017:Apoptosis Regulatory Proteins; D001374:Azacitidine / Q000494:pharmacology*; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D018899:CpG Islands / Q000235:genetics; D019175:DNA Methylation; D015870:Gene Expression / Q000187:drug effects; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000235:genetics / Q000378:metabolism*; D009101:Multiple Myeloma / Q000235:genetics / Q000378:metabolism / Q000473:pathology*; D009363:Neoplasm Proteins / Q000235:genetics / Q000378:metabolism*
D048868:Adaptor Proteins, Signal Transducing; D051017:Apoptosis Regulatory Proteins; D047908:Intracellular Signaling Peptides and Proteins; D009363:Neoplasm Proteins; C417551:XAF1 protein, human; D001374:Azacitidine