The Generation of Stable Oxidative Stress-Resistant Phenotypes in Chinese Hamster Fibroblasts Chronically
3.5. Characterization of O 2 -Resistant Cells
1. In order to quantitate the O2-resistant phenotype of HA1 cells chronically exposed to hyperoxia, the cell lines (desig-nated O2R95 and O2R95-P21%O2) are tested in acute 95%
O2 challenge experiments (compared to HA1 controls), and assayed for maintenance of reproductive integrity using the clonogenic cell survival assay (12, 40).
2. Because O2-resistant cells in culture that are fed fresh medium daily do not experience cell death due to O2 toxi-city, the acute O2 challenge experiments are done in unfed HA1 control and O2-resistant cultures exposed to 95%
Generation of Oxidative Stress-Resistant Phenotypes 191
O2/5% CO2 in humidified 37◦C incubation and surviv-ing fraction ussurviv-ing clonogenic cell survival assay plotted as a function of time in hyperoxia (12, 40).
3. Both the controls and the O2-resistant cells must be at the same cell density (cell number/dish) at the beginning of acute 95% O2 exposure because O2 toxicity in this model system has been shown to be highly cell density dependent (10). This method of doing the acute 95% O2 challenge experiments and expressing the data has been shown to eliminate the artifacts associated with cell density and was used to characterize the cross-resistance of H2O2-resistant cells to O2toxicity (10).
4. To accomplish the acute O2 challenge experiments, place 150,000 cells from each cell line into 60 mm tissue cul-ture plates in 4 mL of complete medium and grow them asynchronously at 37◦C incubation in 5% CO2 for 2 d at which time there are approximately 1,000,000 exponen-tially growing cells per dish in every group.
5. Feed control HA1 cells and O2-resistant cell lines with fresh medium and move the cultures to 95% O2/5% CO2
incubation at 37◦C with no further feeding.
6. Sham-treated cultures in an ambient O2 incubator from each cell line are also included as controls.
7. Remove the cells from hyperoxia after various times of exposure to 95% O2 (24–96 h), rinsed with 4 mL of phosphate-buffered saline, trypsinize, and suspend in 4 mL complete medium, serially dilute in two successive 1:10 dilutions with complete medium.
8. The cells in the originally trypsinized dish are considered to be the 01 dilution and are at a concentration of approx-imately 200,000–400,000 cells/mL, the cells in the first 1:10 dilution are considered to be the 02 dilution and are at a concentration of approximately 20,000–40,000 cells/mL, and the cells in the second 1:10 dilution are con-sidered to be the 03 dilution and are at a concentration of approximately 2,000–4,000 cells/mL.
9. At this point, place 1 mL of the 02 dilution into 9 mL of isoton (Coulter counting solution) and count on a Coul-ter counCoul-ter. Then increasing numbers of cells depend-ing on the severity of the acute 95% O2 challenge dose (approximately 200 for control-untreated HA1 cells or HA1 cells treated with 24 h 95% O2, 200–500 for 48 h O2-treated HA1 cells, 2,000–10,000 for 60 h O2-treated HA1 cells, or 10,000–200,000 for ≥72 h O2-treated HA1 cells) are placed into each of three cloning dishes (60 mm) containing 4 mL of complete media sitting on a
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stainless steel incubator tray and spread evenly on the bot-tom of the cloning dish by vertically and horizontally slid-ing the tray gently on the counter top in the tissue cul-ture hood. O2-resistant cells challenged with hyperoxia are plated in cloning dishes at lower cell densities (usually only 200–500 total) since they are much more resistant to the acute 95% O2 challenge (12). If there is a question as to the survival level of the treated cells (for instance the first time the experiment is accomplished) it is better to plate more dilutions (200–200,000) from each treated dish to ensure that at least one set of cloning dishes will have the required number of colonies to be counted easily (20–200 colonies/dish).
10. Once the cells are evenly spread, the cloning dishes are gen-tly placed into a cloning incubator at 37◦C and 5% CO2for 9–10 d to allow surviving clones to develop. Care is taken not to disturb the cloning dishes during the cloning period to avoid the formation of satellite colonies.
11. At the end of the cloning period the dishes are gently removed from the incubator, rinsed once with 2 mL of 70%
EtOH, and stained 5 min with 2–3 mL of Coomassie blue stain. Colonies are counted under a dissecting microscope and only colonies with at least 50 cells are scored as sur-vivors.
12. Once the colony counts are obtained, surviving fractions for treated and untreated dishes from each cell line are cal-culated by the following expression: colonies counted from each cloning dish/cells plated in the cloning dish = sur-viving fraction. The sursur-viving fraction of untreated dishes from each cell line represents the plating efficiency of that cell line and is used to normalize the calculation of the sur-viving fraction from the O2 treatments using the follow-ing expression: survivfollow-ing fraction of the cells exposed to a given amount of time at 95% O2/surviving fraction of the untreated cells from the same cell line at the same time point= normalized surviving fraction for the given cell line and time of O2exposure (12).
13. The data are then plotted on a log/linear scale with the normalized surviving fraction on the y-axis (log10scale) and exposure time in 95% O2 on the x-axis.
14. Once full survival curves for the entire cell lines are obtained, the resistance of the cell lines can be compared using a calculation of dose modifying factors at 50% iso-survival (DMF50%). DMF50% can be calculated by the fol-lowing expression: time of 95% O2 exposure to reach 50%
survival in the resistant cell line/time to reach 50% survival
Generation of Oxidative Stress-Resistant Phenotypes 193
in the HA1 parental cell line= DMF50% for that particular O2-resistant cell line (12). When this analysis was applied to the HA1 and O2R95-P21%O2cell lines DMFs of 2–3 were obtained and essentially all the HA1 cells were clonogeni-cally inactivated at 96 h of 95% O2, while approximately 50% of the O2R95-P21%O2 cell line survived at this time point (12). This method of doing acute O2 challenge of cells for the purpose of characterizing mechanisms associ-ated with the O2-resistant phenotype has been utilized in a number of publications (12, 18–20, 40) and cell lines selected for O2 resistance derived from HA1 have been shown to maintain a stable resistant phenotype for at least 75 d following the selection protocol (12).
15. Many further studies of cell biological and biochemical characteristics of these O2-resistant cell lines have been accomplished (12, 18–21, 23, 25, 40), and highlights of those studies will be briefly summarized. The O2-resistant cells were approximately the same size, had approximately the same protein/cell, demonstrated slower growth rates (relative to HA1) while being passaged in the feeding pro-tocol in 95% O2 (28 h vs. 14 h) as well as while being passaged in ambient O2 (18 h vs. 14 h), demonstrated increased catalase activity, and demonstrated no apparent increase in ploidy (12, 21). Gross chromosomal rearrange-ments were noted in the O2 selected cells as well as cata-lase gene amplification (that was not seen in HA1 popu-lations exposed to one severe O2 challenge dose) showing that chronic exposure to oxidative stress mediated by 95%
O2 was capable of inducing genomic instability and gene amplification in mammalian cells (21).
16. In addition to these cell biology changes, many biochemi-cal changes were noted in O2-resistant cells. In addition to catalase activity, glutathione peroxidase activity, glutathione transferase activity, CuZn superoxide dismutase activity, MnSOD activity, heme oxygenase activity, aldehyde reduc-tase activity, total glutathione content, and the expression of several stress-responsive genes including GADD153, c-Jun, and a UV-resistance-associated gene (AA490771) were all found to be increased in O2-resistant cells, indi-cating a global response to chronic oxidative stress (20, 12, 18, 23). Using various chemical inhibitors catalase, heme oxygenase, and total glutathione were all found to signifi-cantly contribute to the O2-resistant phenotype (20, 40).
17. In addition, the O2-resistant cells were also found to be cross-resistant to cell killing induced by aldehydic byprod-ucts of lipid peroxidation, hydrogen peroxide, and xanthine oxidase indicating significant overlap between mechanisms
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of resistance to oxidative stress induced by these agents (12, 18, 20, 40). Finally, the O2-resistant cells were found to be resistant to mitochondrial DNA damage induced by expo-sure to xanthine oxidase-mediated oxidative stress as well as demonstrating enhanced mitochondrial DNA damage repair and increased levels of APE activity (25). Overall, these O2-resistant cell lines provide a valuable resource for studying mechanisms of oxidative stress resistance in mam-malian cells. However, it is again worth noting that O2 -resistance mechanisms that arise following chronic expo-sure to hyperoxia are likely to vary with the cell types being studied at the level of both tissue origin and species, so many patterns of resistant phenotypes and mechanisms of resistance are likely to be obtained (41–44).
3.6. Cell Freezing