Sharma M, Jain R, Ionescu E, Darnowski J W
Department of Biophysics, Roswell Park Cancer Institute, Buffalo, New York 14263, USA.
Bioconjug Chem. 1995 Sep-Oct;6(5):536-40. doi: 10.1021/bc00035a006.
We have previously reported that 3'-azido-3'-deoxythymidine (AZT) can possess significant antineoplastic activity in vitro and in vivo when combined with agents which inhibit de novo thymidylate synthesis. Under these conditions cytotoxicity is closely associated with the degree to which AZT is incorporated into DNA. We now report a fluorescence postlabeling technique by which AZT incorporation into DNA can be quantitated without employing radiolabeled AZT. Cultured human colon tumor (HCT-8) cells were exposed to various concentrations of AZT alone and in combination with 5-fluorouracil (FUra). Control cells received the same amount of medium. DNA was isolated from harvested cell pellets (2 x 10(7)). Enzymatic digestion of DNA to the mononucleotide level followed by HPLC analysis of the digest showed that the DNA preparation was free of RNA contamination. The DNA digest was conjugated with dansyl chloride in situ via the phosphoramidate derivative with ethylenediamine. HPLC analysis of the postlabeled nucleotides using fluorescence detection detected 105, 245, and 479 fmol of 5'-monophosphate of AZT (AZTMP) per microg of DNA from cells exposed to 20, 50, and 100 microM AZT, respectively. FUra (3 microM) doubled the AZT incorporation per microg of DNA in cells exposed to 50 and 100 microM AZT. These findings generally support our previously reported data which quantitated (3H)AZT incorporation into cellular DNA and are discussed in light of the potential clinical utility of this technique in assessing the relationship between AZT incorporation into DNA and therapeutic action.
