3-Deazaguanine (3-DG), a
purine analogue, has unusual antitumor activity against experimental mammary
tumor models and a number of other solid
tumors. Others have shown that mutant CHO cells deficient in
hypoxanthine guanine phosphoribosyl
transferase (HGPRTase) or
adenine phosphoribosyl
transferase (
APRTase) are resistant to 3-DG. We developed a L1210 cell line resistant to 3-DG, L1210/3-DG, by subculturing the parent L1210/0 cells in the presence of increasing concentrations of 3-DG. The IC50 was 3.5 microM and 620 microM for L1210/0 and L1210/3-DG, respectively. Cytotoxicity studies proved the resistance to be stable. Examination of the baseline-specific activity of HGPRTase and
APRTase showed that the former was 118-fold lower in L1210/3-DG than in L1210/0, and the latter demonstrated no difference. A 4-h treatment of the cell lines at IC50 doses showed 48% and 23% reductions in
IMP dehydrogenase in L1210/0 and L1210/3-DG, respectively. The rate of de novo
purine biosynthesis was studied by using [14C]
formic acid.
Formate flux increased 2-fold in L1210/3DG in concert with the observed deficiency of HGPRTase in the cell line. 3-DG uptake was studied with [14C]-labelled compound. The total radioactivity was 9-fold higher in L1210/0 than in L1210/3-DG at 2 h. Subsequent chromatographic separation of radioactivity showed the 3-DG and
3-deazaguanosine pools of the drug to be equal in both lines. However, 3-DG
nucleotide pools at 1 min and 2 h were 2.5-fold and 16-fold lower, respectively, in L1210/3-DG than in L1210/0. 3-DG incorporation studies with radiolabelled drug demonstrated that
3-deazaguanine is incorporated in the
acid-insoluble fraction of the cell. These studies conclude that HGPRTase, and not
APRTase, is required for the activation of drug. Inhibition of
IMP dehydrogenase is partially responsible for antitumor activity of the drug. The incorporation of drug into
nucleic acids may be a major mechanism for its antitumor activity. Further studies using a cloned
cDNA probe for
hypoxanthine guanine phosphoribosyltransferase (
HGPRT) demonstrated no change in the
DNA arrangements of the L1210/3-DG cell line, and Northern blot analysis showed approximately equal expression of
mRNA in both cell lines.