Detection Of Arsenic Trioxide Induced P16 Gene Demethylation And Expression In The Multiple Myeloma Cell Line U266

Objective To detect the methylated status of the multiple myeloma cell line U266by the nested-methylation specific PCR + DNA sequence analysis and restrictedendonucleases PCR(REP-PCR). To investigate the mechanism of ArsenicTrioxide(As₂O₃) induced p16 gene demethylation and to introduce a sensitive andspecific method PCR-based for rapid analysis of the promoter methylation status .Method Methylation of the U266 cell line was detected by the nested-methylationspecific PCR (n-MSP) + DNA sequence analysis and restricted endonucleasesPCR(REP-PCR), the expression of p16 gene,DNA methyltransferase 1(DNMT 1),DNMT3A and 3B gene mRNA were determined by RT-PCR, the induced growthinhibition of U266 cell by growth curve and MTT, the DNA content of U266 cell wasanalyzed by flow cytometry after exposed by As₂O₃. Results (1) All cytosines inCpG dinucleotides in untreated U266 cell with As₂O₃ as C, while all cytosines intreated U266 cells have been converted to thymidine. (2) p16 gene fail to express inU266 cell line after methylation. As compared with the β-actin , The expression ofU266 cell p16 gene mRNA was increased to ( 0.22±0.10),(0.59±0.11),(0.68±0.09) after exposed to 0.5umol/L,1.0umol/L and 2.0umol/L As₂O₃ for 72h. (3)As₂O₃ could significantly down-regulate DNA methyltransferase 1(DNMT 1),DNMT3A and 3B gene on mRNA level in a dose dependent manner.(4) U266 cellsline grew slowly and arrested at G0-G1 phrase after treatment with three differentconcentration As2O3.Conclusion As2O3 could activate the expression of p16gene by demethylation or/and by inhibiting DNMT 1,DNMT3A and 3B gene; n-MSP+ DNA sequence analysis is a simple,sensitive and specific method for rapid analysisof the promoter methylation status.