MUTATION-RATES AND MECHANISMS OF RESISTANCE TO ETOPOSIDE DETERMINED FROM FLUCTUATION ANALYSIS

Background: The major known mechanisms of resistance to etoposide incl ude 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 infor mation regarding the distribution, frequency, and origin of these mech anisms in cancer cells. Purpose: We performed fluctuation analysis exp eriments with the human sarcoma cell line, MES-SA, to assess 1) if sel ection or induction mechanisms are involved in resistance to etoposide , 2) mutation rates for cellular resistance to etoposide, and 3) the n ature of the single-step selected surviving clones. Methods: Three gro ups of 10 flasks were seeded with more than 2000 cells each and allowe d 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 mu M (group A), 1.0 mu M (group B), and 5.0 mu M (group C). Surviving colonies in each of the 30 populations were scor ed 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) (g roup C) per cell generation. Of 61 propagated colonies, four of 26 fro m group A, five of 19 from group B, and none of 16 from group C were s tably resistant. Analysis of variance supported the hypothesis of spon taneous mutations rather than induction, conferring etoposide resistan ce in groups A and B. Five of the stably resistant clones were cross-r esistant to doxorubicin. Analysis by polymerase chain reaction failed to detect the expression of the multidrug-resistant gene mdr1 messenge r RNA (mRNA) in any of the clones. No increase in expression of the MR P gene was observed. However, a significant decrease in both Topo II a lpha and II beta mRNA (30%-70%) was found in six of seven stably resis tant 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 mec hanisms of resistance. The experimental design has derived a set of re sistant mutants from a single-step selection. In those clones, decreas ed expression of Topo II is the predominant mechanism selected. Implic ations: These findings suggest that stable resistance to etoposide che motherapy may be acquired by selection of spontaneously arising mutant s rather than induction by drug exposure. The stably resistant clones may represent descendants from a single mutational event in each popul ation.