The cytotoxicity of the 10mer ODN FdUMP[10] towards human colorectal tumor cells was evaluated using a clonogenic assay. FdUMP[10] was more than 100-fold more active than 5-FU at inhibiting colony formation of H630 cells. FdUMP[10] was also evaluated for cytotoxicity in the NCI 60 cell line screen, and showed markedly improved activity relative to 5-FU against numerous tumor cell lines. The in-vivo tolerance of FdUMP[10] is more than three-fold greater per mole fluorinated pyrimidine, than 5-FU.

Cytarabine is a potent anticancer drug that interferes with elongation of the lagging strand at the replication fork during DNA synthesis. The effects of cytarabine substitution on the structural and thermodynamic properties of a model Okazaki fragment were investigated using UV hyperchromicity and 1H NMR spectroscopy to determine how cytarabine alters the physicochemical properties of Okazaki fragments that are intermediates during DNA replication. Two model Okazaki fragments were prepared corresponding to a primary initiation site for DNA replication in the SV40 viral genome. One model Okazaki fragment consisted of five ribo- and seven deoxyribonucleotides on the hybrid strand, together with its complementary (DNA) strand. The second model Okazaki fragment was identical to the first with the exception of cytarabine substitution for deoxycytidine at the third DNA nucleotide of the hybrid strand. Thermodynamic parameters for the duplex to single strand transition for each model Okazaki fragment were calculated from the concentration dependence of the T m at 260 nm. Cytarabine significantly decreased the stability of this model Okazaki fragment, decreasing the melting temperature from 46.8 to 42.4 degrees C at a concentration of 1.33 x 10(-5) M. The free energy for the duplex to single strand transition was 1.2 kcal/mol less favorable for the cytarabine-substituted Okazaki fragment relative to the control at 37 degrees C. Analysis of the temperature dependence of the imino1H resonances for the two duplexes demonstrated that cytarabine specifically destabilized the DNA:DNA duplex portion of the model Okazaki fragment. These results are consistent with inhibition of lagging strand DNA synthesis by cytarabine substitution resulting from destabilization of the DNA:DNA duplex portion of Okazaki fragments in vivo .

The efficacy of treatment with 5-Fluorouracil (5-FU) is limited, in part, by its inefficient conversion to 5-Fluoro-2'-deoxyuridine-5'-O-monophosphate (FdUMP). We present data indicating that FdUMP[10], designed as a pro-drug for intracellular release of FdUMP, is cytotoxic as a consequence of uptake of the multimeric form. FdUMP[10] is stable in cell culture medium, with more than one-half of the material persisting as multimers of at least six nucleotides after a 48 h incubation at 37 degrees C. FdUMP[10] is more than 400 times more cytotoxic than 5-FU towards human colorectal tumor cells (H630). FdUMP[10] also has decreased toxicity in vivo, with doses as high as 200 mg/kg/day (qdx3) administered to Balb/c mice without morbidity, compared to a maximum tolerated dose of 45 mg/kg/day for 5-FU using the same protocol. FdUMP[10] shows reduced sensitivity to OPRTase- and TK-mediated drug resistance, relative to 5-FU and FdU, respectively, and is much more cytotoxic than 5-FU towards cells that overexpress thymidylate synthase. Thus, FdUMP[10] is less susceptible to resistance mechanisms that limit the clinical utility of 5-FU. The increased cytotoxicity, decreased toxicity in vivo, and reduced sensitivity to drug resistance of FdUMP[10], relative to 5-FU, indicates multimeric FdUMP is potentially valuable as an anti-neoplastic agent, either as a single agent, or in combination with 5-FU.

The effects of cytarabine on the structural and thermodynamic properties of an Okazaki fragment were investigated using UV hyperchromicity and 2D 1H NMR. Cytarabine significantly decreased the stability of this model Okazaki fragment, decreasing the melting temperature from 46.8 degrees C to 42.4 degrees C at 1.33 x 10(-5) M. Cytarabine also markedly increased the bend angle of the Okazaki fragment duplex from 20 degrees to 42 degrees. Changes to the structures and stabilities of Okazaki fragments may cause the biological effects of cytarabine.