Woynarowski J M, Napier C, Koester S K, Chen S F, Troyer D, Chapman W, MacDonald J R
Cancer Therapy and Research Center, San Antonio, TX 78245-3217, USA.
Biochem Pharmacol. 1997 Dec 1;54(11):1181-93. doi: 10.1016/s0006-2952(97)00321-3.
6-Hydroxymethylacylfulvene (HMAF, MGI 114) is a new alkylating antitumor sesquiterpenoid with promising and often curative antitumor activity in vivo. This study examined the ability of the drug to damage cellular DNA, induce apoptosis, and affect the cell cycle of CEM human leukemia cells. No bifunctional lesions, interstrand DNA cross-links or DNA-protein cross-links were seen (by alkaline sedimentation and K+/SDS precipitation, respectively) when using up to 50 microM HMAF. The drug possibly formed some monoadducts, as DNA from drug-treated cells impeded primer extension by Taq polymerase, although only partial inhibition was seen even at 200 microM HMAF. HMAF also induced secondary lesions in cellular DNA, single-strand breaks that were detectable (by nucleoid sedimentation and alkaline sucrose gradient analysis) after a 4-hr treatment at HMAF levels as low as 2 microM, comparable to the growth inhibition IC50 value (1.7 microM). A post-treatment incubation of cells in drug-free medium generated substantial amounts of DNA double-stranded fragments of several kbp, suggesting apoptotic fragmentation (>30% of total DNA following treatment with 20 microM HMAF and a 17-hr post-treatment incubation). Chromatin condensation (by ultrastructural analysis) and induction of sub-G1 particles and apoptotic strand breakage (by multiparametric flow cytometry) confirmed induction of apoptosis by HMAF. HMAF preferentially inhibited DNA synthesis (IC50 approximately 2 microM), which is consistent with an S phase block, observed by cell cycle analysis. The pattern of apoptotic DNA fragmentation, inhibition of DNA synthesis, and blockage in the S phase suggests that these events play a role in the antiproliferative activity of HMAF.
6-羟甲基酰基富烯（HMAF，MGI 114）是一种新型烷基化抗肿瘤倍半萜，在体内具有显著且常具治愈性的抗肿瘤活性。本研究检测了该药物损伤细胞DNA、诱导凋亡以及影响CEM人白血病细胞周期的能力。使用高达50微摩尔的HMAF时，未观察到双功能损伤、链间DNA交联或DNA-蛋白质交联（分别通过碱性沉降和K⁺/十二烷基硫酸钠沉淀法）。该药物可能形成了一些单加合物，因为来自药物处理细胞的DNA阻碍了Taq聚合酶的引物延伸，尽管即使在200微摩尔的HMAF浓度下也仅观察到部分抑制。HMAF还诱导细胞DNA产生二级损伤，即单链断裂，在低至2微摩尔的HMAF水平下处理4小时后即可检测到（通过核小体沉降和碱性蔗糖梯度分析），这与生长抑制IC50值（1.7微摩尔）相当。在无药物培养基中对细胞进行处理后孵育产生了大量几千碱基对的DNA双链片段，表明发生了凋亡性片段化（用20微摩尔HMAF处理并进行17小时处理后孵育后，总DNA的>30%）。染色质浓缩（通过超微结构分析）以及亚G1颗粒的诱导和凋亡性链断裂（通过多参数流式细胞术）证实了HMAF诱导凋亡。HMAF优先抑制DNA合成（IC50约为2微摩尔），这与细胞周期分析观察到的S期阻滞一致。凋亡性DNA片段化、DNA合成抑制和S期阻滞的模式表明，这些事件在HMAF的抗增殖活性中起作用。
