Intracellular expression of mini-chaperone confers resistance to apoptosis from oxidative and thermal stress in cos7 cells

Abstract

Purpose : 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.

Methods : 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 37^oC 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 55^oC 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 37^oC 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.

Results : 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.

Conclusions : 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.