Drug uptake and cellular targets of hydroxymethylacylfulvene (HMAF)

Hydroxymethylacylfulvene (HMAF, MGI 114) is a novel antitumor drug and a po tent pro apoptotic agent that has the potential to alkylate cellular nucleo philes. The objective of these studies was to characterize drug uptake and cellular targets far drug binding in human leukemia CEM cells. The uptake o f [C-14]HMAF had two components: a rapid phase (0-10 min) and a slow phase. At 10 mu M 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% b y low temperature (4 degrees) at 4 hr Cell-associated HMAF localized to nuc lear (50%), cytoplasmic (37%), and membrane fractions (10%). Continued drug uptake appeared to be driven by covalent binding to cellular macromolecule s. Approximately 1/4 and 2/3 of cell-associated HMAF formed covalent adduct s after 10 min and 4 hr, respectively, as found by perchloric acid precipit ation. Drug adducts were not readily reversible; 77% of the covalently boun d radiolabel was retained by the cells 20 hr after drug treatment. Combinat ions of DNase, RNase, and proteinase K with perchloric acid precipitation s howed that approximately 60, 30, and 10% of the covalently bound drug was a ssociated with the protein, DNA, and RNA fractions, respectively. Incubatio n-of 100 mu M [C-14]HMAF (24 hr) with purified DNA, serum albumin, thioredo xin, and thioredoxin reductase resulted. in 6, 22, 14, and 11 pmol [C-14]HM AF/mu g DNA or protein, respectively. Results indicate that multiple target s for HMAF binding may contribute to the pro-apoptotic and antiproliferativ e action of the drug. (C) 1999 Elsevier Science Inc.