Further characterization of acquired-resistance to Cisplatin in a rat ovarian tumor-cell line

The proliferation of a rat ovarian tumor cell line (O-342) was completely inhibited by a 2 h exposure to 20 muM cisplatin (DDP) in vitro up to 120 h after its removal, while in its DDP resistant subline (O-342/DDP), the same treatment only caused a transient growth inhibition within the first 24 h post the exposure, followed by the recovery of proliferation at a similar rate as the control cells. DNA interstrand cross links (ISCL) were maximally formed 12 h post DDP treatment in either O-342 or O-342/DDP cells, with a 2.8-fold increase in the sensitive cells at this time (262 vs. 95 rad eq.). After further 12 h incubation, however, 75% of DNA-ISCL was removed in O-342/DDP cells, while only 22% of them were repaired in O-342 cells. DNA single strand breaks (SSB) were produced to a similar extent in both lines but reached a maximum at 12 and 24 h in the resistant and the sensitive cells, respectively. ADP-ribosyl transferase (ADPRT) activity, a DNA repair-associated enzyme, was 2.6-fold higher in O-342/DDP cells compared to the sensitive subline. Following DDP treatment, the activity was stimulated in the sensitive cells with a maximum of about 1.5-fold at 24 h, whereas the inhibitory effect was observed in the resistant cells, although at 12 h it recovered almost to the control level. Flow cytometric analysis showed that there were at least two sub-populations (2n and 4n) in O-342 cells, while only 2n population was observed in O-342/DDP cells. Following DDP exposure, O-342/DDP cells progressed through the cell cycle with only a small and transient accumulation of cells in S-phase at 12 h and 24 h, while in the sensitive cells, it was impossible to distinguish cell-cycle distribution at 12 h due to severe damage, at 24 h most of the cells became arrested in G2-phases, which persisted until the end of the observation (48 h). Our results suggest that both reduced interaction of cellular DNA with DDP and increased DNA repair are contributing factors for development of DDP resistance, which might be directly or indirectly subsequent to alterations of poly (ADP-ribose) metabolism in the resistant cells.