The mechanism of 2'-C-cyano-2'-deoxy-1-beta-D-arabino-pentofuranosylcytosine (
CNDAC) action was investigated in human lymphoblastoid CEM cells and myeloblastic
leukemia ML-1 cells.
CNDAC was metabolized to its 5'-triphosphate and incorporated into
DNA, which was associated with inhibition of
DNA synthesis. After incubation of cells with [(3)H]
CNDAC, metabolites were detected in 3'-->5' phosphodiester linkage and at the 3' terminus of cellular
DNA. Specific enzymatic hydrolysis of
DNA demonstrated that the parent
nucleoside and its 2'-epimer 2'-C-cyano-2'-deoxy-2-ribo-pentofuranosylcytosine accounted for approximately 65% of the total analogs incorporated into
DNA and essentially all of the
drug in the 3'-->5' phosphodiester linkage. In contrast, all detectable radioactivity at 3' termini was associated with 2'-C-cyano-2',3'-didehydro-2',3'-dideoxycytidine. This de facto
DNA chain-terminating
nucleotide arises from an electronic characteristic and cleavage of the 3'-phosphodiester bond subsequent to the addition of a
nucleotide to the incorporated
CNDAC moiety by beta-elimination, a process that generates a single strand break in
DNA. Investigation of the
biological consequences of these actions indicated that, after incubation with
cytostatic concentrations of
CNDAC, cell cycle progression was delayed during S phase, but that cells arrested predominantly in the G(2) phase. This differed from the S phase-arresting actions of
ara-C and
gemcitabine, other
deoxycytidine analogs that inhibit DNA replication but do not cause strand breaks. Thus, once incorporated into
DNA, the
CNDAC molecule appears to act by a dual mechanism that 1) delays the progress of further DNA replication, but 2) upon addition of a deoxynucleotide results in the conversion of the incorporated analog to a de facto
DNA chain terminator at the 3' terminus of a single strand break. It is likely that
DNA strand breaks trigger cell cycle arrest in G(2).