Herzig M C, Arnett B, MacDonald J R, Woynarowski J M
Cancer Therapy and Research Center, Institute for Drug Development, San Antonio, TX 78245-3217, USA.
Biochem Pharmacol. 1999 Jul 15;58(2):217-25. doi: 10.1016/s0006-2952(99)00085-4.
Hydroxymethylacylfulvene (HMAF, MGI 114) is a novel antitumor drug and a potent pro-apoptotic agent that has the potential to alkylate cellular nucleophiles. The objective of these studies was to characterize drug uptake and cellular targets for drug binding in human leukemia CEM cells. The uptake of [14C]HMAF had two components: a rapid phase (0-10 min) and a slow phase. At 10 microM drug (37 degrees), the rapid and slower phase amounted to 0.86 and 0.13 pmol/min/10(6)cells, respectively. HMAF uptake was inhibited 82% by low temperature (4 degrees) at 4 hr. Cell-associated HMAF localized to nuclear (50%), cytoplasmic (37%), and membrane fractions (10%). Continued drug uptake appeared to be driven by covalent binding to cellular macromolecules. Approximately 1/4 and 2/3 of cell-associated HMAF formed covalent adducts after 10 min and 4 hr, respectively, as found by perchloric acid precipitation. Drug adducts were not readily reversible; 77% of the covalently bound radiolabel was retained by the cells 20 hr after drug treatment. Combinations of DNase, RNase, and proteinase K with perchloric acid precipitation showed that approximately 60, 30, and 10% of the covalently bound drug was associated with the protein, DNA, and RNA fractions, respectively. Incubation of 100 microM [14C]HMAF (24 hr) with purified DNA, serum albumin, thioredoxin, and thioredoxin reductase resulted in 6, 22, 14, and 11 pmol [14C]HMAF/microg DNA or protein, respectively. Results indicate that multiple targets for HMAF binding may contribute to the pro-apoptotic and antiproliferative action of the drug.
羟甲基酰基富烯（HMAF，MGI 114）是一种新型抗肿瘤药物，也是一种强效促凋亡剂，具有使细胞亲核试剂烷基化的潜力。这些研究的目的是表征人白血病CEM细胞中药物摄取情况以及药物结合的细胞靶点。[14C]HMAF的摄取有两个成分：快速相（0 - 10分钟）和缓慢相。在10微摩尔药物（37摄氏度）时，快速相和缓慢相分别为0.86和0.13皮摩尔/分钟/10(6)个细胞。4小时时，低温（4摄氏度）可使HMAF摄取抑制82%。细胞相关的HMAF定位于细胞核（50%）、细胞质（37%）和膜部分（10%）。持续的药物摄取似乎是由与细胞大分子的共价结合驱动的。如通过高氯酸沉淀法所发现的，细胞相关的HMAF在10分钟和4小时后分别约有1/4和2/3形成了共价加合物。药物加合物不易逆转；药物处理20小时后，77%的共价结合放射性标记物仍保留在细胞中。脱氧核糖核酸酶、核糖核酸酶和蛋白酶K与高氯酸沉淀法相结合表明，共价结合药物的大约60%、30%和10%分别与蛋白质、DNA和RNA部分相关。100微摩尔[14C]HMAF（24小时）与纯化的DNA、血清白蛋白、硫氧还蛋白和硫氧还蛋白还原酶一起孵育，结果分别为6、22、14和11皮摩尔[14C]HMAF/微克DNA或蛋白质。结果表明，HMAF结合的多个靶点可能有助于该药物的促凋亡和抗增殖作用。
