Effects of 2-chloro-9-(2-deoxy-2-fluoro-beta-D-arabinofuranosyl)adenine on K562 cellular metabolism and the inhibition of human ribonucleotide reductase and DNA polymerases by its 5'-triphosphate.
2-Chloro-9-(2-deoxy-2-fluoro-beta-D-arabinofuranosyl)-adenine (Cl-F-ara-A) has activity against the P388 tumor in mice on several different schedules. Biochemical studies with a chronic myelogenous leukemia cell line (K562) grown in cell culture have been done in order to better understand its mechanism of action. Cl-F-ara-A was a potent inhibitor of K562 cell growth. Only 5 nM inhibited K562 cell growth by 50% after 72 h of continuous incubation. The 5'-triphosphate of Cl-F-ara-A was detected by strong anion exchange chromatography of the acid-soluble extract of K562 cells incubated with Cl-F-ara-A. Competition studies with natural nucleosides suggested that deoxycytidine kinase was the enzyme responsible for the metabolism to the monophosphate. Incubation of K562 cells for 4 h with 50 nM Cl-F-ara-A inhibited the incorporation of [3H]thymidine into the DNA by 50%. Incubation with 0.1, 1, or 10 microM Cl-F-ara-A for 4 h depressed dATP, dCTP, and dGTP pools but did not affect TTP pools. Similar inhibition of deoxyribonucleoside triphosphate pools was seen after incubation with 2-chloro-2'-deoxyadenosine. Both Cl-F-ara-ATP and Cl-dATP potently inhibited the reduction of ADP to dADP in crude extracts of K562 cells (concentration producing 50% inhibition, 65 nM). The effect of Cl-F-ara-ATP on human DNA polymerases alpha, beta, and gamma isolated from K562 cells grown in culture was determined and compared with those of Cl-dATP and 9-beta-D-arabinofuranosyl-2-fluoroadenine triphosphate (F-ara-ATP). Cl-F-ara-ATP was a potent inhibitor of DNA polymerase alpha. Inhibition of DNA polymerase alpha was competitive with respect to dATP (Ki of 1 microM). The three analogue triphosphates were incorporated into the DNA by DNA polymerase alpha as efficiently as dATP. The incorporation of Cl-F-ara-AMP inhibited the further elongation of the DNA chain, similarly to that seen after the incorporation of F-ara-AMP. Extension of the DNA chain after the incorporation of Cl-dAMP was not inhibited as much as it was with either Cl-F-ara-AMP or F-ara-AMP. Cl-F-ara-ATP was not a potent inhibitor of DNA polymerase beta, DNA polymerase gamma, or DNA primase.(ABSTRACT TRUNCATED AT 400 WORDS)
Parker
W B
WB
Kettering-Meyer Laboratory, Southern Research Institute, Birmingham, Alabama 35205.
Shaddix
S C
SC
Chang
C H
CH
White
E L
EL
Rose
L M
LM
Brockman
R W
RW
Shortnacy
A T
AT
Montgomery
J A
JA
Secrist
J A
JA
Bennett
L L
LL
D000227:Adenine Nucleotides; D000255:Adenosine Triphosphate / Q000378:metabolism; D001087:Arabinonucleosides / Q000378:metabolism / Q000494:pharmacology*; D002455:Cell Division / Q000187:drug effects; D000077866:Clofarabine; D003570:Cytidine Triphosphate / Q000378:metabolism; D004247:DNA / Q000096:biosynthesis*; D003841:Deoxycytidine / Q000378:metabolism; D019791:Guanidine; D006146:Guanidines / Q000378:metabolism; D006801:Humans; D019384:Nucleic Acid Synthesis Inhibitors; D012313:RNA / Q000096:biosynthesis; D012264:Ribonucleotide Reductases / Q000037:antagonists & inhibitors*; D014407:Tumor Cells, Cultured
D000227:Adenine Nucleotides; D001087:Arabinonucleosides; D006146:Guanidines; D019384:Nucleic Acid Synthesis Inhibitors; D003841:Deoxycytidine; D012313:RNA; D003570:Cytidine Triphosphate; D000077866:Clofarabine; D000255:Adenosine Triphosphate; D004247:DNA; D012264:Ribonucleotide Reductases; D019791:Guanidine