Validation of 18F-fluorothymidine (FLT) as in vivo molecular probe for measurement of tumor DNA repair synthesis

Objectives FLT has been validated as in vivo marker of tumor cell proliferation. We validated herein its use for a novel application: measurement of tumor DNA repair synthesis.

Methods To measure DNA repair synthesis unconfounded by proliferation-related replicative DNA synthesis, hydroxyurea (HU), known to both inhibit cell proliferation and trigger DNA repair, was used. Cultured actively growing A549 lung adenocarcinoma cells were continuously exposed to 0 or 0.5 mM HU for 72h, a concentration shown to arrest tumor cell growth between 48 and 72h post HU exposure. HU-containing media was removed, cells rinsed and media containing ~10 µCi/ml of ¹⁸FLT added for 60 min. Cells were washed, harvested and cellular FLT uptake, cell number, viability and cell-cycle distribution determined.

Results Cell viability was similar in the HU-exposed and control cells (80% vs. 88%). FLT uptake per A549 cell at 72h post HU exposure was higher than that of control cells (0.0025 vs. 0.0016 cpm/cell) despite only a 1.4-fold increase in cell number during the 72-h incubation period vs. a 2.8-fold increase in control cells. %HU-exposed cells in S-phase of cell cycle was ~3x higher than in control cells (63% vs. 22%) indicating intra-S cycle arrest of HU-exposed cells and that increased cellular FLT uptake is due to intra-S DNA repair synthesis.

Conclusions Our data validate use of FLT for measuring DNA repair in vitro and in vivo after treatment with chemotherapeutics known to induce cell cycle arrest with cessation of replicative DNA synthesis (e.g., HU, camptothecin, gemcitabine). This approach could allow assessment of resistance to drugs caused by enhanced tumor DNA repair.