DNA mismatch repair （MMR） processes the chemically induced mispairs following treatment with clinically important nucleoside analogs such as 6-thioguanine （6-TG） and 5-fluorouracil （5-FU）. MMR processing of thes...DNA mismatch repair （MMR） processes the chemically induced mispairs following treatment with clinically important nucleoside analogs such as 6-thioguanine （6-TG） and 5-fluorouracil （5-FU）. MMR processing of these drugs has been implicated in activation of a prolonged G2/M cell cycle arrest for repair and later induction of apoptosis and/or autophagy for irreparable DNA damage. In this study, we investigated the role of Bcl2 and adenovirus EIB Nineteen-kilodalton Interacting Protein （BNIP3） in the activation of autophagy, and the temporal relationship between a G2/M cell cycle arrest and the activation of BNIP3-mediated autophagy following MMR processing of 6-TG and 5-FU. We found that BNIP3 protein levels are upregulated in a MLHI （MMR＋）-dependent manner following 6-TG and 5-FU treatment. Subsequent small-interfering RNA （siRNA）-mediated BNIP3 knockdown abrogates 6-TG- induced autophagy. We also found that p53 knockdown or inhibition of mTOR activity by rapamycin cotreatment impairs 6-TG- and 5-FU-induced upregulation of BNIP3 protein levels and autophagy. Furthermore, suppression of Checkpoint kinase 1 （Chkl） expression with a subsequent reduction in 6-TG-induced G2/M cell cycle arrest by Chkl siRNA promotes the extent of 6-TG-induced autophagy. These findings suggest that BNIP3 mediates 6-TG- and 5-FU-induced autophagy in a p53- and mTOR-dependent manner. Additionally, the duration of Chkl-activated G2/ M cell cycle arrest determines the level of autophagy following MMR processing of these nucleoside analogs.展开更多
文摘DNA mismatch repair （MMR） processes the chemically induced mispairs following treatment with clinically important nucleoside analogs such as 6-thioguanine （6-TG） and 5-fluorouracil （5-FU）. MMR processing of these drugs has been implicated in activation of a prolonged G2/M cell cycle arrest for repair and later induction of apoptosis and/or autophagy for irreparable DNA damage. In this study, we investigated the role of Bcl2 and adenovirus EIB Nineteen-kilodalton Interacting Protein （BNIP3） in the activation of autophagy, and the temporal relationship between a G2/M cell cycle arrest and the activation of BNIP3-mediated autophagy following MMR processing of 6-TG and 5-FU. We found that BNIP3 protein levels are upregulated in a MLHI （MMR＋）-dependent manner following 6-TG and 5-FU treatment. Subsequent small-interfering RNA （siRNA）-mediated BNIP3 knockdown abrogates 6-TG- induced autophagy. We also found that p53 knockdown or inhibition of mTOR activity by rapamycin cotreatment impairs 6-TG- and 5-FU-induced upregulation of BNIP3 protein levels and autophagy. Furthermore, suppression of Checkpoint kinase 1 （Chkl） expression with a subsequent reduction in 6-TG-induced G2/M cell cycle arrest by Chkl siRNA promotes the extent of 6-TG-induced autophagy. These findings suggest that BNIP3 mediates 6-TG- and 5-FU-induced autophagy in a p53- and mTOR-dependent manner. Additionally, the duration of Chkl-activated G2/ M cell cycle arrest determines the level of autophagy following MMR processing of these nucleoside analogs.
