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Dan-Ning Hu, Ian Chan, Min Chen, Steven A. McCormick, Joseph Walsh; Rescue Of Cybrids Containing Leber Hereditary Optic Neuropathy (LHON) Mutation Using Bax-inhibiting Peptide. Invest. Ophthalmol. Vis. Sci. 2011;52(14):5440.
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© ARVO (1962-2015); The Authors (2016-present)
To investigate the use of Bax-inhibiting peptide V5 (BIP) to block apoptosis in cybrids containing the LHON mutation. BIP is derived from the amino acid sequence of the Bax-binding domain of Ku70 and blocks the Bax-Bak oligomerization on outer mitochondria membranes.
Homeoplasmic 143B osteosarcoma cybrids containing the mtDNA (G11778A) mutation were grown in Dulbecco’s Modified Eagle Medium (DMEM) with high glucose supplemented with 10% fetal bovine serum. Cells were seeded into 96 well plates and cultured with DMEM, glucose-free galactose medium (GFGM) and GFGM with BIP at 10, 30, 100 and 300 µmol concentrations. After 48 hrs, 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT, 50 µl of 1 mg/ml) was added, incubated for 4 hrs, and replaced by DMSO. A microplate spectrophotometer at 540 nm was used to evaluate cell survival.
Compared to cybrids in DMEM medium, cell viability from MTT assay showed that cybrid survival % dropped to 34% after incubation in GFGM after 48 hours. Adding BIP to GFGM at 10, 30, 100 and 300 µmol concentrations increased survival in a dose dependent fashion to 42%, 48%, 50% and 56%, respectively, after 48 hrs. P-values for 2-tailed t-test of cell survival for BIP at concentrations of 10, 30, 100 and 300 µmol with GFGM vs. GFGM alone were 0.02, 0.01, 0.0002 and 0.0001 respectively.
BIP demonstrated statistically significant dose-dependent protective effects in the LHON cybrid model.
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