ETOPOSIDE CAUSES ILLEGITIMATE V(D)J RECOMBINATION IN HUMAN LYMPHOID LEUKEMIC-CELLS

Etoposide is one of the most widely used antineoplastics. Unfortunatel y, the same treatment schedules associated with impressive efficacy ar e associated with an increased risk of secondary acute myeloid leukemi a (AML), which has prompted its withdrawal from some treatment regimen s, thereby potentially compromising efficacy against the original tumo r. Because etoposide-associated AML is characterized by site-specific illegitimate DNA recombination, we studied whether etoposide could dir ectly cause site-specific deletions of exons 2 and 3 in the hprt gene, Human lymphoid CCRF-CEM cells were treated with etoposide for 4 hours , and DNA was isolated after subculturing. The deletion of exons 2 and 3 from hprt was assayed by a quantitative polymerase chain reaction ( PCR) method. In the absence of etoposide treatment, the frequency of d eletions of exons 2 and 3 was very low (5.05 x 10(-8)). After exposure to 10 mu mol/L etoposide, the frequency of the exon 2+3 deletion was increased immediately after and at 24 hours after etoposide treatment (65 to 89 x 10(-8)) and increased to higher levels (128 to 173 x 10(-8 )) after 2 and 6 days of subculture (P<.001 overall). The frequency of the exon 2+3 deletion assessed at 6 days of subculture after 4 hours of 0, 0.25, 1, 2.5, 5, and 10 mu mol/L etoposide treatment increased w ith etoposide concentration, is, 5.05 x 10(-8), 89.2 x 10(-8) 108 x 10 (-8), 142 x 10(-8), 163 x 10(-8), and 173 x 10(-8), respectively (P<.0 001). Sequencing of a subset of amplified products confirmed the prese nce of DNA sequences at the breakpoints consistent with V(D)J recombin ation. By contrast, exon 2+3 deletions after etoposide treatment in th e myeloid cell lines KG-1A and K562 showed no evidence of V(D)J recomb inase in their genesis. We conclude that etoposide can induce the ille gitimate site-specific action of V(D)J recombinase on an unnatural DNA substrate after a single treatment in human lymphoid cells. (C) 1996 by The American Society of Hematology.