Effects on DNA integrity and apoptosis induction by a novel antitumor sesquiterpene drug, 6-hydroxymethylacylfulvene (HMAF, MGI 114)

Jan M. Woynarowski, Cheryl Napier, Steven K. Koester, Shih Fong Chen, Dean Troyer, William Chapman, John R. MacDonald

Research output: Contribution to journalArticlepeer-review

92 Scopus citations

Abstract

Hydroxymethylacylfulvene (HMAF, MGI 114) is a new alkylating antitumor sesquiterpenoid with promising and often curative antitumor activity in due. 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 lip to 50 μM HMAF. The drug possibly formed some monoadducts, as DNA from drug-treated cells impeded primer extension by Tag polymerase, although only partial inhibition was seen even at 200 μM 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 μM, comparable to the growth inhibition IC50 value (1.7 μM). 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 μM HMAF and a 17-hr post-treatment incubation). Chromatin condensation (by ultrastructural analysis) and induction of sub-G, particles and apoptotic strand breakage (by multiparametric flow cytometry) confirmed induction of apoptosis by HMAF. HMAF preferentially inhibited DNA synthesis (IC50 ~ 2 μM), 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.

Original languageEnglish (US)
Pages (from-to)1181-1193
Number of pages13
JournalBiochemical Pharmacology
Volume54
Issue number11
DOIs
StatePublished - Dec 1 1997

Keywords

  • 6-hydroxymethylacylfulvene
  • Apoptosis
  • Cytotoxic activity
  • DNA fragmentation
  • S phase block

ASJC Scopus subject areas

  • Biochemistry
  • Pharmacology

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