Effects on DNA integrity and apoptosis induction by a novel antitumor sesquiterpene drug, 6-hydroxymethylacylfulvene (HMAF, MGI 114).
6-Hydroxymethylacylfulvene (HMAF, MGI 114) is a new alkylating antitumor sesquiterpenoid with promising and often curative antitumor activity in vivo. 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 up to 50 microM HMAF. The drug possibly formed some monoadducts, as DNA from drug-treated cells impeded primer extension by Taq polymerase, although only partial inhibition was seen even at 200 microM 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 microM, comparable to the growth inhibition IC50 value (1.7 microM). 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 microM HMAF and a 17-hr post-treatment incubation). Chromatin condensation (by ultrastructural analysis) and induction of sub-G1 particles and apoptotic strand breakage (by multiparametric flow cytometry) confirmed induction of apoptosis by HMAF. HMAF preferentially inhibited DNA synthesis (IC50 approximately 2 microM), 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.
Woynarowski
J M
JM
Cancer Therapy and Research Center, San Antonio, TX 78245-3217, USA. jan@jerry.iddw.saci.org
Napier
C
C
Koester
S K
SK
Chen
S F
SF
Troyer
D
D
Chapman
W
W
MacDonald
J R
JR
D000970:Antineoplastic Agents / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects*; D002453:Cell Cycle / Q000187:drug effects; D004247:DNA / Q000096:biosynthesis / Q000187:drug effects*; D004249:DNA Damage; D053938:DNA Fragmentation; D005434:Flow Cytometry; D006801:Humans; D012717:Sesquiterpenes / Q000494:pharmacology*
D000970:Antineoplastic Agents; D012717:Sesquiterpenes; C102714:irofulven; D004247:DNA