Effects of 1-beta-D-arabinofuranosylcytosine on DNA replication intermediates monitored by pH-step alkaline elution.
The pH-step alkaline elution method enables the isolation and quantification of nascent DNA (nDNA) replication intermediates, including Okazaki fragments, short length nDNA from replicon origins, longer lengths of nascent but subgenomic length nDNA (molecular weight, 20-30 x 10(6)), and full (or genomic) length nDNA (L. C. Erickson et al., Chromosoma, 74: 125-139, 1979). We utilized this technique to study, in HL-60 cells, the effects of 1-beta-D-arabinofuranosylcytosine (ara-C) on the formation of these replication intermediates and the kinetics of transit of radiolabel from [3H]thymidine ([3H]dThd) or [3H]-ara-C through these nDNA fragments and into full length nDNA. In the continuous presence of [3H]-ara-C (4 microM), the majority of radiolabel (greater than 85%) remained in the nascent subgenomic fractions, with 30-50% remaining in Okazaki fragments. These proportions did not change substantially with increasing time of exposure to [3H]-ara-C (from 1 to 24 h), although the total amount of [3H]-ara-C incorporated into DNA continued to increase with increasing time of exposure. In contrast, when cells were exposed to [3H]-ara-C for 1 h, placed in drug-free medium, and studied by the pH-step method at various times thereafter, the transit of radiolabel through progressively larger nDNA intermediates and into full length nDNA was rapid and equal to that of [3H]dThd in cells not exposed to drug. The observed elution of [3H]-ara-C in the subgenomic-length DNA fragments was not due to ara-C-induced breaks in template (parental) DNA and subsequent incorporation of [3H]-ara-C into the template strand, since ara-C treatment of cells prelabeled with [14C]dThd failed to cause substantial elution of the 14C label at the various pH steps used. In studies of the effects of ara-C on [3H]dThd incorporation into nDNA, concentrations of 1 to 10 microM ara-C inhibited total incorporation of radiolabel into DNA by greater than 90% and incorporation into full length nDNA by greater than 97%. In contrast, these concentrations of ara-C failed to decrease the amount of [3H]dThd incorporated into Okazaki fragments or other non-mitochondrial low molecular weight nDNA, compared to control. These studies demonstrate that, in HL-60 cells, ara-C causes profound inhibition of nascent chain elongation, does not cause chain termination, and does not inhibit initiation. In fact, ara-C may stimulate initiation, leading credence to recent theories proposing endoreduplication or reinitiation as consequences of ara-C incorporation into DNA.
Ross
D D
DD
Division of Developmental Therapeutics, University of Maryland Cancer Center, Baltimore 21201.
Chen
S R
SR
Cuddy
D P
DP
D001088:Arabinonucleotides / Q000378:metabolism; D003561:Cytarabine / Q000378:metabolism / Q000494:pharmacology*; D003568:Cytidine Monophosphate / Q000031:analogs & derivatives / Q000378:metabolism; D004261:DNA Replication / Q000187:drug effects*; D004273:DNA, Neoplasm / Q000096:biosynthesis; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D015473:Leukemia, Promyelocytic, Acute / Q000378:metabolism; D013936:Thymidine / Q000378:metabolism; D014407:Tumor Cells, Cultured
D001088:Arabinonucleotides; D004273:DNA, Neoplasm; D003561:Cytarabine; C024888:1-beta-D-arabinofuranosylcytosine 5'-monophosphate; D003568:Cytidine Monophosphate; D013936:Thymidine