Protection of acute myeloblastic leukemia cells against apoptotic cell death by high glutathione and gamma-glutamylcysteine synthetase levels during etoposide-induced oxidative stress

Background: Etoposide mediates its cytotoxicity by inducing apoptosis. Thus , mechanisms which regulate apoptosis should also affect drug resistance. O xidants and antioxidants have been shown to participate in the regulation o f apoptosis. We were interested in studying whether responsiveness of acute myeloblastic leukemia (AML) cells to etoposide is mediated by oxidative st ress and glutathione levels. Patients and methods: Two subclones of the OCI/AML-2 cell line which are et oposide-sensitive (ES), and etoposide-resistant (ER), were established by t he authors at the University of Oulu, and used as models. Assays for apopto sis included externalization of phosphatidylserine (as evidenced by annexin V binding), and caspase activation as indicated by cleavage of poly(ADP-ri bose)polymerase (Western blotting). Peroxide formation was analyzed by flow cytometry. Glutathione and gamma-glutamylcysteine synthetase (gamma-GCS) l evels were determined spectrophotometrically and by Western blotting, respe ctively. Results: Etoposide-induced apoptosis was evident 12 hours after treatment i n the ES subclone, but was apparent in the ER subclone only after 24 hours. The basal glutathione and gamma-GCS levels were higher in the ER than the ES subclone. Etoposide increased peroxide formation in both subclones after 12-hour exposure. Significant depletion of glutathione was observed in the ES subclone during etoposide exposure, while glutathione levels were maint ained in the ER subclone. In neither of the subclones was induction of gamm a-GCS observed during 24-hour exposure to etoposide. Furthermore, the catal ytic subunit of gamma-GCS was cleaved during apoptosis, concurrent with dep letion of intracellular glutathione. When glutathione was depleted by treat ment with buthionine sulfoximine, a direct inhibitor of gamma-GCS, the sens itivity to etoposide was increased, particularly in the ER subclone. Conclusions: The results underline the significance of glutathione biosynth esis in the responsiveness of AML cells to etoposide. The molecular mechani sms mediating glutathione depletion during etoposide exposure might include the cleavage of the catalytic subunit of gamma-GCS.