25-hydroxycholesterol induces the differential expression of manganese
superoxide dismutase and the apurinic/apyrimidinic endonuclease 1 in the human lymphoblastic leukemic CEM cells. Nicole Villafañe*, María del Rocío Castro† and Sylvette Ayala-Torres†. *University of Puerto Rico Río Piedras Campus and †University of Puerto Rico Medical Sciences Campus.
Oxysterols represent a vast family of natural cholesterol oxidation products that are potent inducers of apoptosis in leukemic lymphoid cells. The mechanism responsible for this process is unknown, but it has been suggested that increased generation of mitochondrial-generated reactive oxygen species may play an important role in the induction of lymphoid apoptosis. Previous work in our laboratory indicates that 25-hydroxycholesterol (25OHC), a potent oxysterol, induces apoptosis of human lymphoid leukemic CEM cells via the induction of mitochondrial DNA (mtDNA) damage. We hypothesize that 25OHC-induced apoptosis involves the regulation of the expression of two enzymes that localize to mitochondria: manganese superoxide dismutase (MnSOD) and the apurinic/apyrimidinic (AP) endonuclease 1 (APE1). MnSOD is an antioxidant protein that scavenges the superoxide anion and APE1 is involved in the repair of oxidative mtDNA damage. To test our hypothesis, we performed time course and dose-response studies after exposure of oxysterol-sensitive (CEMC7) and oxysterol-resistant (M10R5) cell clones to 25OHC and measured the expression of MnSOD and APE1 by Western blot analysis. Our results show a ~50% decrease in the expression of MnSOD in the oxysterol-sensitive CEMC7 cells at 24 and 48h, whereas levels of MnSOD expression increased in the oxysterol-resistant M10R5 cells only 48h after treatment with 1000 nM 25OHC. Treatment with 300 and 1000 nM 25OHC induced a statistically significant ~20% decrease in the expression of APE1 after 24 and 48h in the oxysterol-sensitive CEMC7 cells, while no significant changes were observed in the expression of APE1 in the oxysterol-resistant M10R5 cells. We conclude that 25OHC-induced apoptosis involves the differential expression of MnSOD and APE1, suggesting that both the antioxidant and repair pathways may play a role in the sensitivity of CEMC7 leukemic cells to undergo apoptosis. 25OHC may be a good candidate as a chemotherapeutic agent. Supported by the Amgen Bio-Minds Program.
