Inhibition of primer RNA formation in CCRF-CEM leukemia cells by fludarabine triphosphate.
The effects of fludarabine triphosphate (Fara-ATP), 1-beta-D-arabinofuranosylcytosine 5'-triphosphate (ara-CTP), and aphidicolin on primer RNA and DNA synthesis in human CCRF-CEM leukemia cells were investigated. RNA-primed Okazaki fragment synthesis was monitored by first incubating whole cell lysates for 10 min in the presence or absence of the compound and then following the incorporation of [alpha-32P]ATP and [3H]dTTP into the primer RNA and DNA portions, respectively, of the Okazaki fragments. In whole cell lysates the degree of DNA synthesis inhibition induced by Fara-ATP was directly related to the extent of primer RNA synthesis inhibition over the entire range of Fara-ATP concentrations tested (10-50 microM). In contrast, primer RNA formation was stimulated by concentrations of ara-CTP (25-200 microM) and aphidicolin (0.5-5 micrograms/ml) that inhibited DNA synthesis. The primer RNA recovered from cell lysates incubated with either Fara-ATP, ara-CTP, or aphidicolin was of normal length, predominately 11 nucleotides. Fara-ATP was a more potent inhibitor of the polydeoxythymidylate primase activity than of the DNA polymerase alpha/delta activities present in the 100,000 x g supernatants of CCRF-CEM cells. Fara-ATP was a noncompetitive inhibitor of DNA primase with respect to ATP [50% inhibitory concentration, 2.3 +/- 0.3 (SD) microM, Ki = 6.1 +/- 0.3 (SE) microM] and the Km(ATP)/Ki (Fara-ATP) was 25. The 50% inhibitory concentration values of Fara-ATP for DNA polymerases alpha/delta activities on calf thymus DNA were 43 +/- 1.6 (SD) microM and greater than 100 microM with respect to dATP and dTTP. The effects of ara-CTP and aphidicolin on these enzymes were opposite those seen with Fara-ATP, since 50% inhibitory concentrations of either ara-CTP or aphidicolin for DNA polymerases alpha/delta did not inhibit polydeoxythymidylate primase activity. The results provide evidence that fludarabine phosphate blocks DNA synthesis in CCRF-CEM cells through inhibition of primer RNA formation. In contrast, the accumulation of primer RNA and RNA-primed Okazaki fragments that is induced by ara-CTP and aphidicolin could lead to the rereplication and amplification of chromosomal DNA segments.
Catapano
C V
CV
Department of Biochemistry, Bowman Gray School of Medicine, Wake Forest University, Winston-Salem, North Carolina 27103.
Chandler
K B
KB
Fernandes
D J
DJ
D016590:Aphidicolin; D001085:Arabinofuranosylcytosine Triphosphate / Q000494:pharmacology; D019915:DNA Primase; D004273:DNA, Neoplasm / Q000096:biosynthesis*; D004259:DNA-Directed DNA Polymerase / Q000096:biosynthesis*; D003848:Deoxyguanine Nucleotides / Q000494:pharmacology; D004224:Diterpenes / Q000494:pharmacology; D006801:Humans; D007938:Leukemia / Q000378:metabolism*; D012313:RNA / Q000096:biosynthesis*; D012316:RNA Nucleotidyltransferases / Q000096:biosynthesis*; D014407:Tumor Cells, Cultured; D001084:Vidarabine Phosphate / Q000031:analogs & derivatives* / Q000494:pharmacology
D004273:DNA, Neoplasm; D003848:Deoxyguanine Nucleotides; D004224:Diterpenes; C034477:RNA primers; D001084:Vidarabine Phosphate; D001085:Arabinofuranosylcytosine Triphosphate; C042382:fludarabine phosphate; C000101:ara-ATP; D016590:Aphidicolin; D012313:RNA; C040364:N(2)-(4-n-butylphenyl)-2'-deoxyguanosine 5'-triphosphate; D019915:DNA Primase; D012316:RNA Nucleotidyltransferases; D004259:DNA-Directed DNA Polymerase