Drug uptake and cellular targets of hydroxymethylacylfulvene (HMAF)
Autor: | John R. MacDonald, Jan M. Woynarowski, Brenda Arnett, Maryanne C. Herzig |
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Rok vydání: | 1999 |
Předmět: |
Macromolecular Substances
RNase P Thioredoxin reductase Serum albumin Antineoplastic Agents Thymus Gland Biochemistry Tumor Cells Cultured medicine Humans Carbon Radioisotopes Pharmacology Dose-Response Relationship Drug biology Chemistry Serum Albumin Bovine Biological activity DNA Neoplasm Proteinase K In vitro Mechanism of action biology.protein Thioredoxin medicine.symptom Sesquiterpenes |
Zdroj: | Biochemical Pharmacology. 58:217-225 |
ISSN: | 0006-2952 |
Popis: | 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. |
Databáze: | OpenAIRE |
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