Related Experiment Videos
Characterization of a set of Chinese hamster ovary variant cell lines demonstrating differing sensitivity to
B L Kuehl1, M Buchwald, A M Rauth
1Department of Medical Biophysics, University of Toronto, Ontario, Canada.
Abstract:
Three related Chinese hamster ovary (CHO) cell lines derived from CHO-K1R cells (MMC3-A2, 21-1 and G1B) previously shown to differ in their sensitivity to mitomycin C (MMC), were investigated in more detail to determine the factors controlling this sensitivity. A separately maintained wild type cell line (CHO-K1TOR) was included in this study for comparison. Continuous (chronic) exposure of the five cell lines to MMC during the 10-day colony forming assay demonstrated a 15-fold range in MMC sensitivity between the most sensitive cell line (MMC3-A2) and the most resistant cell line (G1B) with CHO-K1R, 21-1 and CHO-K1TOR falling at intermediate levels. Acute aerobic exposure (0-5 h) to MMC resulted in a reduced fivefold range of sensitivities, which was further reduced to a three-fold range under hypoxic exposure conditions. These results were suggestive of differences in the aerobic enzymatic activation of MMC as a possible mechanism contributing to the varying sensitivities. There was no correlation between the one-electron reducing enzyme NADPH:cytochrome P-450 oxidoreductase (P450R) activity and cellular sensitivity to MMC. The five cell lines had similar levels of reduced glutathione (GSH), suggesting that oxygen homeostasis was not correlated with the cells, differing sensitivity to MMC. A correlation did exist between NAD(P)H:quinone oxidoreductase (DT-diaphorase) activity and cellular sensitivity to MMC under chronic exposure conditions for the cell lines. High DT-diaphorase levels were also correlated with a reduced ability of oxygen to modulate MMC toxicity. Levels of P450R and DT-diaphorase were not altered significantly during five-hour aerobic or hypoxic exposures of control cells.(ABSTRACT TRUNCATED AT 250 WORDS)
Insights
Investigating Chinese hamster ovary (CHO) cell lines revealed that NAD(P)H:quinone oxidoreductase (DT-diaphorase) activity, not NADPH:cytochrome P-450 oxidoreductase (P450R), influences mitomycin C (MMC) sensitivity, particularly under chronic exposure.
Area of Science:
- Cell Biology
- Biochemistry
- Toxicology
Background:
- Chinese hamster ovary (CHO) cell lines exhibit varying sensitivities to mitomycin C (MMC).
- Understanding the factors controlling MMC sensitivity is crucial for cellular research and drug development.
- Previous studies indicated differences in MMC sensitivity among related CHO cell lines.
Purpose of the Study:
- To investigate the underlying mechanisms controlling differential sensitivity to mitomycin C (MMC) in related Chinese hamster ovary (CHO) cell lines.
- To determine the role of specific enzymes, such as NADPH:cytochrome P-450 oxidoreductase (P450R) and NAD(P)H:quinone oxidoreductase (DT-diaphorase), in MMC sensitivity.
- To assess the influence of aerobic and hypoxic conditions on MMC toxicity.
Main Methods:
- Utilized five related CHO cell lines, including CHO-K1R derivatives and a wild-type CHO-K1TOR line.
- Employed a 10-day colony forming assay to assess cellular sensitivity to MMC under continuous exposure.
- Evaluated MMC sensitivity under acute aerobic and hypoxic exposure conditions.
- Measured activities of NADPH:cytochrome P-450 oxidoreductase (P450R) and NAD(P)H:quinone oxidoreductase (DT-diaphorase).
- Assessed levels of reduced glutathione (GSH) to evaluate oxygen homeostasis.
Main Results:
- A 15-fold range in MMC sensitivity was observed among the five CHO cell lines under chronic exposure.
- Acute aerobic and hypoxic exposures reduced the range of MMC sensitivities.
- No correlation was found between P450R activity and MMC sensitivity.
- Similar reduced glutathione (GSH) levels across cell lines indicated oxygen homeostasis was not a primary factor.
- A significant correlation existed between DT-diaphorase activity and MMC sensitivity under chronic exposure.
- High DT-diaphorase levels correlated with reduced oxygen modulation of MMC toxicity.
Conclusions:
- DT-diaphorase activity, not P450R, is a key determinant of MMC sensitivity in CHO cells, especially under chronic exposure.
- Aerobic enzymatic activation of MMC may contribute to varying sensitivities.
- Oxygen's role in modulating MMC toxicity is influenced by DT-diaphorase levels.