Abstract

The mechanism of 2′-C-cyano-2′-deoxy-1-β-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 [³H]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 β-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 G₂ 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 G₂.