Re-expression of p16 gene in the myeloma cell line U266 induced by synergy of sodium butyrate and 5-Aza-2'-deoxycytidine.

OBJECTIVE

To understand the synergic effect of histone deacetylase inhibitor sodium butyrate (SB) and demethylating agent 5-Aza-2'- deoxycytidine (5-Aza-CdR) on cell growth and to explore the possibility of re-expression of the hypermethylated and silenced p16 gene in the myeloma cell line U266.

METHODS

U266 cells were cultured in RPMI 1640 in the presence of varied doses of SB and 5-Aza-CdR, and the growth curve was obtained by trypan-blue exclusion assay and cell count. The cell cycle was analyzed by flow cytometry and the expression level of mRNA and protein of p16 gene were detected by reverse transcriptase-PCR and Western blotting, respectively.

RESULTS

The cell growth was arrested by treatment with 5-Aza-CdR alone or SB alone. Increased inhibition effect was shown in synergic treatment of SB and 5-Aza-CdR. The G(1) phase of cell cycle was arrested by 5-Aza-CdR combined with SB, which, however, did not occur when SB or 5-Aza-CdR was used alone. 5-Aza-CdR alone induced the expression of p16 gene in a concentration-dependent manner, whereas SB alone only induced its low-level expression. The expression level of both mRNA and protein of p16 gene was increased significantly by synergic application of SB and 5-Aza-CdR.

CONCLUSION

Hypermethylated and silenced p16 gene in U266 cell line can be markedly reactivated by synergic treatment with demethylating agent 5-Aza-CdR and histone deacetylase inhibitor SB, and the cell growth can be inhibited and cell cycle arrested at G(1) phase.