Chen C L, Fuscoe J C, Liu Q, Relling M V
Department of Pharmaceutical Sciences, St Jude Children's Research Hospital, Memphis, TN 38101-0318, USA.
Blood. 1996 Sep 15;88(6):2210-8.
Etoposide is one of the most widely used antineoplastics. Unfortunately, the same treatment schedules associated with impressive efficacy are associated with an increased risk of secondary acute myeloid leukemia (AML), which has prompted its withdrawal from some treatment regimens, thereby potentially compromising efficacy against the original tumor. Because etoposide-associated AML is characterized by site-specific illegitimate DNA recombination, we studied whether etoposide could directly 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 deletions of exons 2 and 3 was very low (5.05 x 10(-8)). After exposure to 10 mumol/ 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 mumol/L etoposide treatment increased with etoposide concentration, ie, 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 < .0001). Sequencing of a subset of amplified products confirmed the presence of DNA sequences at the breakpoints consistent with V(D)J recombination. By contrast, exon 2 + 3 deletions after etoposide treatment in the myeloid cell lines KG-1A and K562 showed no evidence of V(D)J recombinase in their genesis. We conclude that etoposide can induce the illegitimate site-specific action of V(D)J recombinase on an unnatural DNA substrate after a single treatment in human lymphoid cells.
依托泊苷是使用最广泛的抗肿瘤药物之一。不幸的是，与显著疗效相关的相同治疗方案会增加继发性急性髓系白血病（AML）的风险，这促使其从某些治疗方案中撤出，从而可能影响对原发肿瘤的疗效。由于依托泊苷相关的AML以位点特异性非法DNA重组为特征，我们研究了依托泊苷是否能直接导致hprt基因外显子2和3的位点特异性缺失。用人淋巴细胞CCRF-CEM细胞用依托泊苷处理4小时，传代培养后分离DNA。通过定量聚合酶链反应（PCR）方法检测hprt外显子2和3的缺失情况。在未进行依托泊苷处理时，外显子2和3的缺失频率非常低（5.05×10⁻⁸）。暴露于10μmol/L依托泊苷后，在依托泊苷处理后即刻及24小时，外显子2 + 3缺失频率增加（65至89×10⁻⁸），传代培养2天和6天后增加到更高水平（128至173×10⁻⁸）（总体P <.001）。在0、0.25、1、2.5、5和10μmol/L依托泊苷处理4小时后传代培养6天评估的外显子2 + 3缺失频率随依托泊苷浓度增加，即分别为5.05×10⁻⁸、89.2×10⁻⁸、108×10⁻⁸、142×10⁻⁸、163×10⁻⁸和173×10⁻⁸（P <.0001）。对一部分扩增产物进行测序证实了断点处存在与V(D)J重组一致的DNA序列。相比之下，髓系细胞系KG-1A和K562经依托泊苷处理后的外显子2 + 3缺失在其发生过程中未显示V(D)J重组酶的证据。我们得出结论，依托泊苷在对人淋巴细胞进行单次处理后，可诱导V(D)J重组酶在非天然DNA底物上发生非法的位点特异性作用。
