QUANTITATIVE-ANALYSIS OF 3'-AZIDO-3'-DEOXYTHYMIDINE INCORPORATION INTO DNA IN HUMAN COLON TUMOR CELLS

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. Unde r these conditions cytotoxicity is closely associated with the degree to which AZT is incorporated into DNA. We now report a fluorescence po stlabeling technique by which AZT incorporation into DNA can be quanti tated without employing radiolabeled AZT. Cultured human colon tumor ( HCT-8) cells were exposed to various concentrations of AZT alone and i n combination with 5-fluorouracil (FUra). Control cells received the s ame amount of medium. DNA was isolated from harvested cell pellets (2 x 10(7)). Enzymatic digestion of DNA to the mononucleotide level follo wed 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 ethylenediami ne. HPLC analysis of the postlabeled nucleotides using fluorescence de tection detected 105, 245, and 479 fmol of 5'-monophosphate of AZT (AZ TMP) per mu g of DNA from cells exposed to 20, 50, and 100 mu M AZT, r espectively. FUra (3 mu M) doubled the AZT incorporation per mu g of D NA in cells exposed to 50 and 100 mu M AZT. These findings generally s upport our previously reported data which quantitated (H-3)AZT incorpo ration into cellular DNA and are discussed in light of the potential c linical utility of this technique in assessing the relationship betwee n AZT incorporation into DNA and therapeutic action.