Recovery from potentially lethal damage induced by spirohydantoin mustard on 9L cells in vitro

Spirohydantoin mustard (SHM) has been shown to cross the blood-brain barrier in dogs and has shown activity against mouse brian tumors in vivo and against 9L rat brain tumor cells in vitro. Factors influencing the recovery of monolayer 9L cells from SHM-induced potentially lethal damage were studied. The pattern of recovery was similar in exponentially growing and plateau-phase cultures. Recovery occurred in either Earle's balanced salt solution (EBSS) or depleted medium (DM), and was inhibited in fresh medium (FM). The recovery process had a half-life of approximately 4 hours and was completed within 18 hours. The enhancement of survival was dependent on drug concentration and temperature. The magnitude of recovery decreased as the temperature was lowered and was inhibited at ambient temperature, which suggested that an enzymatic repair process was responsible for recovery. The distribution of cells through the cell cycle after SHM treatment was determined using a flow cytometer. Incubation of exponentially growing and plateau-phase cells in either EBSS or DM impeded the progression of cells from G1 into S phase, but cells incubated in FM progressed into S phase by 24 hours. We conclude that the recovery occurred primarily when cells were in G1 phase and potentially lethal damage would be fixed when cells initiate DNA synthesis within 18 hours after administration of SHM. Therefore, optimal therapeutic benefit may depend on drug scheduling.