Imbalanced DNA synthesis induced by cytosine arabinoside and fludarabine in human leukemia cells.
Previous studies have demonstrated that cytosine arabinoside (araC) induces an accumulation of Okazaki fragments, while fludarabine (FaraA) inhibits Okazaki fragment synthesis. We extended these observations in the present study to provide insights into various mechanisms by which these anticancer drugs affect DNA replication and induce genomic instability in human CEM leukemia cells. Neither araC nor FaraA induced a detectable amount of re-replicated DNA in S-phase cells, which indicated that drug-induced alterations in Okazaki fragment synthesis were not accompanied by DNA re-replication. Synthesis on both leading and lagging DNA strands within the c-myc locus was measured in cells incubated with equitoxic concentrations of araC or FaraA. In araC-treated cells, nascent DNA from the lagging strand was enriched about 5-fold compared with the leading strand. In contrast, FaraA did not induce any replication imbalance. AraC- and FaraA induced changes in the frequency of N-(phosphonacetyl)-l-aspartate (PALA) resistance and the extent of CAD gene amplification were monitored as markers of drug-induced genomic instability. At concentrations that reduced cloning efficiency by 50% (IC(50)), araC increased the frequency of PALA resistance about 4-fold, while FaraA did not have a significant effect on the frequency of PALA resistance. Pretreatment with araC also increased the extent of CAD gene amplification. We propose that the imbalanced DNA synthesis induced by araC leads to the accumulation of Okazaki fragments on the lagging arms and single-stranded DNA regions on the leading arms of replication forks. The formation of these abnormal replication structures was associated with the generation of genomic instability.
Carbone
G M
GM
Department of Experimental Oncology, Hollings Cancer Center, Medical University of South Carolina, 86 Jonathan Lucas Street, P.O. Box 250955, Charleston, SC 29425, USA.
Catapano
C V
CV
Fernandes
D J
DJ
D000970:Antineoplastic Agents / Q000494:pharmacology*; D003561:Cytarabine / Q000494:pharmacology*; D004261:DNA Replication / Q000187:drug effects; D004273:DNA, Neoplasm / Q000096:biosynthesis / Q000187:drug effects*; D006801:Humans; D007938:Leukemia; D014407:Tumor Cells, Cultured; D014740:Vidarabine / Q000031:analogs & derivatives* / Q000494:pharmacology*
D000970:Antineoplastic Agents; D004273:DNA, Neoplasm; D003561:Cytarabine; D014740:Vidarabine; C024352:fludarabine