Drug uptake and cellular targets of hydroxymethylacylfulvene (HMAF)

Maryanne C.S. Herzig, Brenda Arnett, John R. MacDonald, Jan M. Woynarowski

Research output: Contribution to journalArticlepeer-review

48 Scopus citations

Abstract

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 μM drug (37°), the rapid and slower phase amounted to 0.86 and 0.13 pmol/min/106cells, respectively. HMAF uptake was inhibited 82% by low temperature (4°) 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 μM [14C]HMAF (24 hr) with purified DNA, serum albumin, thioredoxin, and thioredoxin reductase resulted in 6, 22, 14, and 11 pmol [14C]HMAF/μg DNA or protein, respectively. Results indicate that multiple targets for HMAF binding may contribute to the pro-apoptotic and antiproliferative action of the drug. Copyright (C) 1999 Elsevier Science Inc.

Original languageEnglish (US)
Pages (from-to)217-225
Number of pages9
JournalBiochemical Pharmacology
Volume58
Issue number2
DOIs
StatePublished - Jul 15 1999

Keywords

  • DNA adducts
  • Drug uptake
  • HMAF
  • Illudins
  • MGI 114
  • Macromolecule binding

ASJC Scopus subject areas

  • Biochemistry
  • Pharmacology

Fingerprint

Dive into the research topics of 'Drug uptake and cellular targets of hydroxymethylacylfulvene (HMAF)'. Together they form a unique fingerprint.

Cite this