Action of (E)-2'-deoxy-2'-(fluoromethylene)cytidine on DNA metabolism: incorporation, excision, and cellular response.
(E)-2'-deoxy-2'-(fluoromethylene)cytidine (FMdC) is a new analog of deoxycytidine with promising anticancer activity. We investigated the action of FMdC on DNA metabolism by evaluating its incorporation into DNA, its excision from DNA in vitro, and the role of the incorporation of FMdC into DNA in causing cytotoxicity. In vitro DNA primer extension demonstrated that FMdC nucleotides were incorporated with relatively high substrate efficiency into the C sites of the elongating DNA strand. Once incorporated, FMdC became a poor substrate for further chain elongation by DNA polymerases, resulting in a termination of DNA synthesis at the sites of incorporation. Furthermore, the 3' --> 5' exonuclease activity of DNA polymerase epsilon or wild-type p53 protein was ineffective in removing the incorporated FMdC from DNA in vitro. FMdC also showed potent cytotoxic activity against human leukemia and solid tumor cells. Incubation with a low concentration of FMdC (10 nM) induced cell cycle arrest at S or G1 phases, but the cells eventually died as the time of incubation increased. Compared with HL-60 cells, human myeloid ML-1 cells with wild-type p53 were more sensitive to FMdC, but the S or G1 phase arrest did not seem to depend on the presence or absence of p53. Inhibiting the incorporation of FMdC into cellular DNA by aphidicolin suppressed the cytotoxic effect of the compound. We conclude that the incorporated FMdC nucleotide profoundly disrupts DNA synthesis and resists excision by exonucleases, and that incorporation of this analog into DNA is a key molecular event responsible for the drug's cytotoxicity.
Zhou
Yan
Y
Department of Experimental Therapeutics, The University of Texas M. D. Anderson Cancer Center, Houston, Texas 77030, USA.
Achanta
Geetha
G
Pelicano
Helene
H
Gandhi
Varsha
V
Plunkett
William
W
Huang
Peng
P
D002453:Cell Cycle / Q000187:drug effects; D004247:DNA / Q000187:drug effects* / Q000378:metabolism; D004256:DNA Polymerase I / Q000037:antagonists & inhibitors; D004257:DNA Polymerase II / Q000378:metabolism*; D003841:Deoxycytidine / Q000031:analogs & derivatives / Q000378:metabolism / Q000494:pharmacology*; D005434:Flow Cytometry; D018922:HL-60 Cells / Q000187:drug effects / Q000378:metabolism; D006801:Humans; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism*
D016159:Tumor Suppressor Protein p53; D003841:Deoxycytidine; D004247:DNA; D004256:DNA Polymerase I; D004257:DNA Polymerase II; C086432:tezacitabine