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Effects of 1-ß-D-Arabinofuranosylcytosine in DNA Replication Intermediates Monitored by pH-Step Alkaline Elution¹

Division of Developmental Therapeutics, Program of Oncology, University of Maryland Cancer Center [D. D. R., D. P. C.], Division of Hematology, Department of Medicine [D. D. R.], and Program in Medical Technology, Department of Pathology [S-R. S. C.], University of Maryland School of Medicine, Baltimore, Maryland 21201

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 10⁶), 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-ß-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 µM), the majority of radiolabel (>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 µM ara-C inhibited total incorporation of radiolabel into DNA by >90% and incorporation into full length nDNA by >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.

¹ Supported in part by Grant RO-1-CA40188 of the NIH, National Cancer Institute. Presented in part to the eightieth annual meeting of the American Association for Cancer Research, San Francisco, CA, May 1989.

² To whom requests for reprints should be addressed, at University of Maryland Cancer Center, Bressler Research Building, Room 9-015, 655 West Baltimore St., Baltimore, MD 21201.

Received 6/ 6/89. Accepted 11/ 6/89.