Mechanism of cis-diamminedichloroplatinum(II)-induced cytotoxicity: role of G2 arrest and DNA double-strand breaks

Abstract

DNA has been implicated as the critical target for cis-diamminedichloroplatinum(II) (cis-DDP)-induced cytotoxicity. In vitro, DNA-platinum adducts inhibit DNA synthesis. An assessment of the inhibition of DNA synthesis in murine leukemia L1210 cells demonstrated that, although cell division was halted, DNA replication continued for a period of time. The DNA underwent almost a complete doubling even in cells that did not divide. Flow cytometric analysis demonstrated a slowed synthetic phase which progressed to a block in the G2 phase of the cell cycle. The duration of the G2 block was proportional to the concentration of cis-DDP. Low concentrations of cis-DDP caused the cells to be transiently blocked in the G2 phase for 24 to 48 h. Higher concentrations of cis-DDP resulted in a G2 arrest that was not reversed by 96 h. After this time, the arrested cells appeared to disintegrate, rather than recover. Cell survival and trypan blue exclusion studies indicated that, at low drug concentrations, cells which had transiently arrested in the G2 phase survived, while at higher concentrations only a limited number of survivors were responsible for the observed recovery of growth. Analysis of DNA double-strand breaks showed that significant numbers of breaks only occurred at concentrations of cis-DDP that subsequently led to debris detectable on the flow cytometer and to loss of trypan blue exclusion. The formation of these breaks appeared to be the first detectable change that was indicative of cell death. It is proposed that cells arrest in the G2 phase because they are unable to transcribe damaged DNA and make mRNA essential for passage into mitosis. DNA repair probably overcomes this arrest. Cell death may therefore be a consequence of the inability to adequately recover transcription.