Figure 5.

Menadione-induced death of Nu-hOGG1 MEFs is accompanied by mitochondrial dysfunction and activation of the calpain pathway. (A) Suppression of mitochondrial degeneration by hOGG1-2a. At least 50 mitochondrial sections of MEFs were examined, and percentages of mitochondria with EDDs are shown. In OGG1-null MEFs, most mitochondria were degenerated and hardly identifiable 24 h after the exposure (‡). Fisher's exact probability test, ^*P<0.05, ^**P<0.01. (B) Depletion of mtDNA in each type of MEF after exposure to menadione. mt-Co1 DNA was amplified from total cellular DNA prepared from MEFs, which were harvested 2–6 h after a 60-min exposure to 50 μM menadione. The intensity of mt-Co1 DNA was normalized to that of nuclear Gapdh DNA, and the relative intensity of each sample to that of the control sample is shown at the bottom of each lane. (C) Time-dependent alterations in the intracellular ATP level, mitochondrial membrane potential, and mitochondrial Ca²⁺ level in each type of MEF after exposure to menadione. The ATP index in each type of MEF after a 60-min exposure to 50 μM menadione was determined, and levels are shown as a percentage of the untreated control (mean±s.e.m., n=6). The mitochondrial membrane potential was monitored by JC-1 and the mitochondrial Ca²⁺ level was monitored with rhod-2. Fluorescence intensities of JC-1 and rhod-2 of 30 and 50 cells, respectively, were examined, and are shown as a percentage of the untreated control (mean±s.e.m.). Top panel, Nu-hOGG1 MEFs; middle panel, Mt-hOGG1 MEFs; bottom panel, Nu/Mt-hOGG1 MEFs. (D) Activation of calpain after exposure to menadione. Calpain activity in a whole-cell extract prepared from OGG1-null (Null), Nu-hOGG1 (Nu), and Mt-hOGG1 MEFs (Mt) was determined 6 h after exposure to menadione (25, 50 μM). Control, no exposure. Relative calpain activities normalized to that of the untreated control for OGG1-null MEFs are shown (mean±s.d., n=3 per experiment). (E) Suppression of cell death by calpain inhibitor. OGG1-null, Nu-hOGG1, and Mt-hOGG1 MEFs were preincubated in the presence or absence of 20 μM MDL28170 (+MDL28170) for 60 min and were exposed to menadione for 60 min. Cell viability at 24 h after the exposure was determined by the trypan blue exclusion test. Results from three independent experiments, each of which was run in triplicate, are presented (mean±s.d.). Student's t-test with or without MDL28170, ^*P<0.05. (F) Suppression of cell death by CsA. OGG1-null, Nu-hOGG1, and Mt-hOGG1 MEFs were preincubated for 60 min in the presence or absence of 100 nM CsA, and then exposed to 50 μM menadione for 60 min. Cell viability at 24 h after the exposure was determined by the trypan blue exclusion test, and results from three independent experiments, each of which was run in triplicate, are presented (mean±s.d.). Student's t-test with or without CsA, ^*P<0.05. (G) Suppression of calpain activation by CsA. Nu-hOGG1 MEFs preincubated for 60 min in the presence or absence of 100 nM CsA were exposed to menadione (25, 50 μM) for 60 min, then calpain activity was determined periodically (mean±s.d., n=3 per experiment). Relative calpain activities normalized to that of an untreated control (open bar) are shown. Student's t-test, ^**P<0.01.