Studies in mouse L-cells on the incorporation of 1-beta-D-arabinofuranosylcytosine into DNA and on inhibition of DNA polymerase by 1-beta-D-arabinofuranosylcytosine 5'-triphosphate.

Abstract

Studies have been carried out in mouse L-cells on the incorporation of 1-β-d-arabinofuranosylcytosine (ara-C) into DNA and on the inhibition of DNA polymerase by the 5′-triphosphate of ara-C (ara-CTP) to determine whether either of the two current models, incorporation into DNA or inhibition of DNA polymerase, could account for ara-C action. With a modification of the McGrath-Williams technique, it was found that ara-C was initially incorporated into small (Okazaki) pieces of DNA but shifted into longer DNA strands when cells were washed and incubated in a medium free of ara-C-3H. On degradation of DNA from ara-C-3H-labeled cells with micrococcal nuclease and spleen phosphodiesterase, it was found that most of the ara-C appeared to be in internucleotide rather than terminal linkages, suggesting that chain elongation is not stopped by the addition of ara-C to a growing strand. Studies on ara-C incorporation into nucleic acids failed to show any correlation between the amount of incorporation and the degree of lethality. With crude extracts of L-cells, it was found that ara-CTP was a competitive inhibitor of DNA polymerase, and values of 9.0 ± 4.3 and 8.7 ± 5.2 × 10-6 m were obtained for the Michaelis-Menten constants of dCTP and ara-CTP, respectively. Calculations based on these values and on measured values of the dCTP and ara-CTP concentrations in vivo indicated that the predicted inhibition of DNA synthesis was significantly smaller than that actually observed in whole cells. Nevertheless, an evaluation of all of the available data suggests that the most plausible model for the action of ara-C is that DNA synthesis is inhibited by inhibition of DNA polymerase.