Cell-cycle progression and response of germ cell tumors to cisplatin in vitro

Testicular germ cell tumors (GCTs) are highly sensitive to cisplatin-based chemotherapy. It has been suggested that the chemosensitivity of GCTs can be partially attributed to the preference of apoptosis induction over a p21-mediated G1/S phase cell-cycle arrest following induction of p53. Since cell-cycle progression can be manipulated by a growing number of targeted agents, a thorough understanding of the impact of cell-cycle progression on drug-induced cell death might help to enhance the efficacy of chemotherapy. The aim of this study was to assess the cell-cycle dependence of cisplatin-induced cell death in an in vitro model of GCTs. Cell-cycle progression and induction of apoptosis were assessed by flow cytometry and Western blot analysis of PARP cleavage in the GCT derived cell lines, NT2 and 2102 EP, and compared with the breast carcinoma cell line MCF-7. Response to treatment was assessed in different phases of the cell cycle after synchronization by serum depletion and contact inhibition. Following cisplatin exposure, unsynchronized cells accumulated in G2/M after 28 h. This arrest was reversible at sublethal cisplatin doses (0.5-4.5 µM for 2 h). At higher concentrations, cells accumulated in G2 and died in G2/M-arrest. A 2-h exposure of cells in G2/M with 10 µM cisplatin resulted in a higher apoptotic index 70 h after treatment (74 and 70% for NT2 and 2102 EP, respectively) compared to treatment in G1/S (34 and 38%). Synchronized cells treated in G1 showed PARP cleavage after 48 h following cisplatin exposure, whereas treatment in G2 resulted in PARP cleavage already after 24 h. Cisplatin-induced cell death in GCTs is highly dependent on cell-cycle phase. All crucial events are restricted to the G2/M phase: cisplatin-induced DNA-damage is sensed, the apoptotic process is initiated and eventually executed in this phase of the cell cycle. The cells are most sensitive to cisplatin in this phase of the cell cycle. As far as the development of targeted agents is concerned, inhibition of the cell cycle in G1/S phase is likely to result in a protective effect against cisplatin, whereas agents arresting cells in G2/M may exert a synergistic effect.