RELATIONSHIP BETWEEN CYTOXICITY AND SITE-SPECIFIC DNA RECOMBINATION AFTER IN-VITRO EXPOSURE OF LEUKEMIA-CELLS TO ETOPOSIDE

Background: Etoposide, an inhibitor of the normal religation activity of the nuclear enzyme topoisomerase II, can induce a secondary acute m yeloid leukemia characterized by site-specific DNA rearrangements. The schedule of drug administration appears to he a clinical risk Patter for this devastating treatment complication. Purpose: We tested the hy pothesis that prolonged exposure of leukemia cells in vitro to low con centrations of etoposide, compared with short exposures to high concen trations, could produce equivalent of greater desired cytotoxic effect s, with decreased occurrence of undesired site-specific double-strande d DNA recombinational events (i.e., recombinogenesis). Methods: We use d the frequency of V(D)J (variable-diversity-joining) recombinase-medi ated deletions of exons 2 and 3 of the hypoxanthine phosphoribosyltran sferase (HPRT) gene as a biomarker of etoposide-induced, nonhomologous , site-specific DNA rearrangement. A polymerase chain reaction-based t echnique was used to measure exon 2 + 3 deletions in human lymphoid le ukemia CCRF-CEM cells 6 days after either 4-hour or 24-hour treatment with etoposide al clinically relevant concentrations. Cytotoxic effect s of etoposide determined by the number of viable cells present in the treated compared with the control [i.e., untreated] cells) were measu red 6 days after treatment of the cells. The frequency of the exon 2 3 deletion following the two treatment-duration conditions was compar ed by use of the Mantel-Haenszel statistic. All P values resulted from two-sided tests. Results: Cytotoxicity increased with increasing etop oside concentration and exposure duration. as expected, By day 6, the frequency of exon 2 + 3 deletions was significantly higher (global P v alue = .0003) after the 4-hour treatment than after the 24-hour treatm ent, regardless of whether the frequency was assessed at etoposide con centrations achieving equivalent (e.g., 95%) cytotoxicity (14.2 x 10(- 7) versus 3.1 x 10(-7)) or at equivalent etoposide concentrations (e.g ., 1 mu M) (10.8 x 10(-7) versus 1.3 x 10(-7)). Thus, the ratio of des ired cytotoxic to undesired recombinogenic effects was higher with the 24-hour schedule, After the treated cells were subcloned at limiting dilutions, the frequency of the exon 2 + 3 deletion increased from 16. 3 x 10(-7) to 4.33 x 10(-3)-indicating that the recombinational event is not necessarily lethal. Conclusion: For all drug concentrations and levels of cytotoxicity studied in CCRF-CEM cells, there was a greater ratio of cytotoxicity to genetic recombination following prolonged ex posure to etoposide than following brief exposure. Implication: These data suggest that recombinogenesis is not inextricably linked to cytot oxicity. If confirmed in the clinical setting, the use of prolonged do sage schedules may provide a means to decrease the risk of etoposide-i nduced acute myeloid leukemia without compromising treatment efficacy.