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Puttur Santhoshkumar, K Krishna Sharma; Intracellular expression of mini-chaperone confers resistance to apoptosis from oxidative and thermal stress in cos7 cells. Invest. Ophthalmol. Vis. Sci. 2017;58(8):4502.
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© ARVO (1962-2015); The Authors (2016-present)
Therapeutic potentials of peptide chaperone derived from αA-crystallin is well established. The objective of this study is to prepare a mini-gene construct capable of expressing functional peptide chaperone(s) in mammalian cells.
A stable cell line expressing a mini-chaperone (MGKFVIFLDVKHFSPEDLTVK) was prepared by transfecting Cos7 cells with linearized pcDNA3.1(+) vector containing the engineered mini-gene inserted between EcoRI and XbaI sites and grown on Geneticin selective medium including 5% serum and maintained at 37oC and 5% CO2. Cos7 cells transfected with linearized empty vector served as control. The anti-apoptotic activity of the intracellularly expressed mini-chaperone was tested by treating the cells grown on a 96-well plate with 75, 150 and 300 µM H2O2 for 16 h followed by TUNEL assay. The anti-apoptotic activity of the mini-chaperone against hyperthermia was investigated by incubating the culture plates at 55oC for 1h and the live/dead cell in the two groups were visualized by staining the cells with HCS NuclearMask™ Red stain and Image-IT® DEAD Green™ Viability Stain at 37oC for 30 min. The plates were imaged using the SpectraMax Minimax 300 Imaging Cytometer (Molecular Devices) and the live/dead cells were quantified using Softmax Pro software.
Cell cultures transfected with mini-gene showed 55 – 80% less TUNEL positive cells when compared to control groups. Cells transfected with empty vector (positive control) failed to provide any protection against H2O2 and was comparable to untransfected cells (negative control). In the Live/Dead assay, cells transfected with empty vector showed increased number of dead cells (8.6 + 2.3%) compared to cells transfected with mini-gene ( 2.2 + 1.2 %). Our data suggests that the intracellular expression of mini-chaperone protects Cos7 cells from oxidative and thermal stress.
Functional mini-chaperones can be expressed in cells. Mini-gene therapy can be a possible strategy for treating diseases involving oxidative stress, protein aggregation, and apoptosis.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.
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