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Inhibition of Primer RNA Formation in CCRF-CEM Leukemia Cells by Fludarabine Triphosphate¹

Department of Biochemistry, Bowman Gray School of Medicine of Wake Forest University, Winston-Salem, North Carolina 27103

The effects of fludarabine triphosphate (Fara-ATP), 1-ß-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 [-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 µM). In contrast, primer RNA formation was stimulated by concentrations of ara-CTP (25–200 µM) and aphidicolin (0.5–5 µg/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 / 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) µM, K_i = 6.1 ± 0.3 (SE) µM] and the K_m(ATP)/K_i (Fara-ATP) was 25. The 50% inhibitory concentration values of Fara-ATP for DNA polymerases / activities on calf thymus DNA were 43 ± 1.6 (SD) µM and > 100 µM 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 / 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.

¹ This work was supported by Grant CH-226 from the American Cancer Society and by USPHS Grant CA-44597 awarded by the National Cancer Institute.

² Supported in part by a Berti Fellowship from Champion Industries, Winston-Salem, NC.

³ Scholar of the Leukemia Society of America, Inc.; to whom requests for reprints should be addressed.

Received 8/20/90. Accepted 1/22/91.

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