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Mutation rates and mechanisms of resistance to etoposide determined from fluctuation analysis
J P Jaffrézou1, K G Chen, G E Durán
1Department of Medicine, Stanford University School of Medicine, Calif.
Background:
The major known mechanisms of resistance to etoposide include altered expression of its target enzyme, topoisomerase II (Topo II), and the multidrug-resistant phenotypes encoded by the mdr1 and MRP (multidrug resistance-associated protein) genes. There is little information regarding the distribution, frequency, and origin of these mechanisms in cancer cells.
Purpose:
We performed fluctuation analysis experiments with the human sarcoma cell line, MES-SA, to assess 1) if selection or induction mechanisms are involved in resistance to etoposide, 2) mutation rates for cellular resistance to etoposide, and 3) the nature of the single-step selected surviving clones.
Methods:
Three groups of 10 flasks were seeded with more than 2000 cells each and allowed to grow to near confluence (approximately 3 x 10(6) cells per flask). After reseeding, each group received etoposide for 1 week at a final concentration of 0.5 microM (group A), 1.0 microM (group B), and 5.0 microM (group C). Surviving colonies in each of the 30 populations were scored and individually harvested.
Results:
Mutation rates were estimated at 2.9 x 10(-6) (group A), 5.7 x 10(-7) (group B), and 1.7 x 10(-7) (group C) per cell generation. Of 61 propagated colonies, four of 26 from group A, five of 19 from group B, and none of 16 from group C were stably resistant. Analysis of variance supported the hypothesis of spontaneous mutations rather than induction, conferring etoposide resistance in groups A and B. Five of the stably resistant clones were cross-resistant to doxorubicin. Analysis by polymerase chain reaction failed to detect the expression of the multidrug-resistant gene mdr1 messenger RNA (mRNA) in any of the clones. No increase in expression of the MRP gene was observed. However, a significant decrease in both Topo II alpha and II beta mRNA (30%-70%) was found in six of seven stably resistant and six of six unstably resistant mutants.
Conclusions:
Our study demonstrates that resistance to etoposide arises spontaneously, with most clones surviving either stochastically or through very labile mechanisms of resistance. The experimental design has derived a set of resistant mutants from a single-step selection. In those clones, decreased expression of Topo II is the predominant mechanism selected.
Implications:
These findings suggest that stable resistance to etoposide chemotherapy may be acquired by selection of spontaneously arising mutants rather than induction by drug exposure. The stably resistant clones may represent descendants from a single mutational event in each population.
Insights
Etoposide resistance in cancer cells arises from spontaneous mutations, not drug induction. Decreased topoisomerase II expression is the primary mechanism in resistant clones, suggesting selection of pre-existing mutants.
Area of Science:
- Cancer Biology
- Pharmacology
- Genetics
Background:
- Etoposide resistance mechanisms include altered topoisomerase II (Topo II) expression and multidrug resistance genes (mdr1, MRP).
- Limited data exists on the prevalence and origin of these resistance mechanisms in cancer cells.
Purpose of the Study:
- Investigate spontaneous mutation rates for etoposide resistance in MES-SA sarcoma cells.
- Determine if selection or induction mechanisms drive etoposide resistance.
- Characterize the resistance mechanisms in single-step selected clones.
Main Methods:
- Fluctuation analysis was performed on MES-SA cells exposed to varying etoposide concentrations (0.5-5.0 µM).
- Mutation rates were calculated, and surviving colonies were isolated and analyzed.
- Gene expression (mdr1, MRP, Topo II alpha/beta mRNA) was assessed using polymerase chain reaction.
Main Results:
- Mutation rates for etoposide resistance ranged from 1.7 x 10⁻⁷ to 2.9 x 10⁻⁶ per cell generation.
- Stable resistance was observed in a subset of clones, supporting spontaneous mutation over induction.
- Decreased Topo II alpha and II beta mRNA levels were detected in most resistant clones, while mdr1 and MRP expression remained unchanged.
Conclusions:
- Etoposide resistance predominantly arises from spontaneous mutations, with decreased Topo II expression as the main selected mechanism.
- Stable resistance appears to result from single mutational events, suggesting selection of pre-existing resistant clones.
- Findings imply that etoposide resistance is acquired through selection of spontaneous mutants rather than drug-induced changes.