M. Sharma et al., QUANTITATIVE-ANALYSIS OF 3'-AZIDO-3'-DEOXYTHYMIDINE INCORPORATION INTO DNA IN HUMAN COLON TUMOR CELLS, Bioconjugate chemistry, 6(5), 1995, pp. 536-540
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.