Mammalian resistance to oxidative stress: a comparative analysis

Abstract

Changes in gene expression represent a major protective mechanism, and enforced overexpression of individual genes has been shown to protect cells. However, no large-scale comparison of genes involved in mammalian oxidative stress protection has yet been described. Using filter microarray and restriction fragment differential display technology, hydrogen peroxide (H2O2)-resistant variants of hamster HA-1 fibroblasts and human HL-60 promyelocytes were found to possess a surprising lack of commonality in specific modulated genes with the single exception of catalase, supporting the hypothesis that catalase overexpression is critical for resistance to H2O2. Comparison of two cell lines from the same species (hamster) selected with an exogenous oxidative stressing agent (H2O2) and an endogenous metabolic oxidative stressing agent (95% O2) also revealed little commonality in modulation of specific mRNAs with the exception of glutathione S-transferase enzymes and catalase. Acute oxidative stress in HL-60 led to the modulation of a limited subset of the genes associated with chronic oxidative stress resistance. Overall, these results suggest that mammalian resistance to oxidative and perhaps other stress does not require a significant number of common genes but rather only a limited number of key genes (e.g., catalase in our model systems) in combination with others that are cell type and stress agent specific.

Figure 1

Example of microarray analysis. Human gene filter arrays containing 5184 noncontrol cDNAs were hybridized with probes obtained from HL-60 control and HP100-resistant cells. The cDNA probes were prepared from 6 μg of extracted total RNA in the presence of ³³P radiolabel. After hybridization and washing, a final wash stringency of 55°C and 0.5× SSC, 1% SDS was employed. The arrays were then phosphoimaged using a Storm 860 phosphoimager with ImageQuant software.

Figure 2

Expression of ubiquitin mRNA in HL-60 cells. Northern blot analysis of ubiquitin, identified by RFDD as a modulated mRNA. Extracted RNAs from HL-60 control, HP50, and HP100 log-phase unstressed cell cultures were electrophoresed, blotted to nylon paper, and then hybridized to radiolabeled ubiquitin cDNA probes specific for each ubiquitin variant indicated. β-Actin was used as a loading control.

Figure 3

Expression of ubiquitin in oxidative stress-resistant human and hamster cells. (A) Western blot analysis of HL-60 versus resistant sublines. 30 μg of protein per lane was electrophoresed on a 15% SDS-polyacrylamide gel, electroblotted, and hybridized to a rabbit polyclonal antibody to bovine ubiquitin. Ubiquitin signal was detected using horse radish peroxidase-conjugated secondary antibody followed by signal development with ECL Plus and signal quantitation with a Storm 860 phosphoimager and ImageQuant software. Results are shown for two different experiments (Exp. 1 and Exp. 2). (B) Northern blot analysis of HA-1 versus OC14 subline. RNA extracted from HA-1 and OC14 cells was prepared, electrophoresed, blotted, and hybridized as above using radiolabeled ubiquitin as a probe.

Figure 4

Northern blot confirmation of RFDD-identified catalase and myleoperoxidase. cDNAs to catalase and myleoperoxidase (MPO) were radiolabeled and used to probe Northern blots containing RNA from HL-60 and HP100 cells. GAPDH was used as a loading control.