Zeitschrift für Wirbelsäulen- und Neurochirurgie

Differential Cell Death Effects in Glioblastoma after Drug- Induced DNA Damage Laboratory Investigation

Chen MY, Fuji T, Cheung B, Graf MR, Holt SE, Clark AJ, Chidambaram A, Lin J, Jandial R and Broaddus WC

Object: Glioblastomas are difficult tumors to eradicate because of resistance to apoptosis and other mechanisms of programmed cell death such as autophagy. We hypothesized that DNA damage, regardless of etiology, would cause autophagy. To test our hypothesis, we examined the ability of two DNA damaging agents, 1, 3-Bis (2-chloroethyl)-1-nitrosourea (BCNU) and cisplatin, to induce autophagy. Materials and methods: DNA damage was assessed by western blot for γ-H2AX and immunofluorescence for phospho-53BP1. Autophagy was measured by microtubule-associated protein 1 light chain (LC3) and beclin 1 western blots, acridine orange staining, response to the 3-MA inhibitor, and autophagosome detection using electron microscopy. To study apoptosis, we examined levels of BAX and BAK, TUNEL staining, inhibition with ZVAD.fmk and caspase 3/7 activation. Results: The levels of the DNA damage indicators γ-H2AX and 53BP1 increased with both BCNU and cisplatin. While LC3-II autophagy proteins were highly expressed in BCNU samples, LC3-II levels were below the limits of detection in cells treated with cisplatin. Caspase 3/7 activation only slightly increased with BCNU, but markedly increased with cisplatin. Surprisingly, BAX and BAK levels did not change in response to either chemotherapeutic compound. Significant TUNEL staining was evident in cisplatin, but not BCNU-treated cells, and the pancaspase inhibitor, ZVAD.fmk, did not diminish cell death after BCNU treatment. Conclusion: Although both drugs caused DNA damage, we concluded not all DNA damage results in a specific type of cell death, as BCNU-related cell death in glioblastomas occurs through autophagy and cisplatin predominantly induces apoptosis. The specific molecular mechanisms underlying the activation of autophagy remain obscure.