Mechanism of in vivo sister-chromatid exchange induction by 5-azacytidine

Abstract

The aim of the present study was to explore the in vivo mechanism of sister-chromatid exchange (SCE) induction by 5-azacytidine (5-azaC) in murine bone marrow cells. Experiments were performed to examine SCE induction in response to different doses of 5-azaC as well as several exposures. Additionally, we examined the persistence of SCE induction and the effect of bromodeoxiuridine (BrdU) incorporation. Sister-chromatid differentiation was obtained by injecting mice intraperitoneally with BrdU absorbed to activated charcoal. Before BrdU injection, different doses of 5-azaC were administered intraperitoneally either singly or in multiples. Colchicine in an aqueous solution was administered subcutaneously 22 h after BrdU injection. Two hours later, animals were sacrificed by cervical dislocation and both femurs were dissected. Bone marrow cells were processed to obtain chromosome preparations, which were stained by the fluorescence plus Giemsa method. Results indicate that 5-azaC caused SCE, albeit to a limited extent. In order to discern whether the limitation was due to cytotoxicity or to partial 5-azaC incorporation, we administered multiple sub-toxic doses of 5-azaC. This treatment increased 5-azaC incorporation and reduced cytotoxicity, but did not raise SCE frequency, indicating that the limitation was not due to either of the two factors mentioned above. SCE frequency induced by 5-azaC persisted for at least eight cell divisions, confirming that this agent had caused inhibition of DNA methyltransferase and subsequently the reduction of DNA re-methylation, which in turn induced the expression of a number of SCE-prone sites. Finally, SCE induction in response to 5-azaC was completely dependent on BrdU incorporation. The data allow us to conclude that 5-azaC causes SCE to a limited extent; limited SCE induction was not due to the direct effect of incorporation or cytotoxicity of 5-azaC, but rather the generation of a number of SCE-prone sites, the expression of which persists for at least eight cell divisions and is dependent on BrdU incorporation.