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

Abstract

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/Marrest. 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.

Role of the pro-survival molecule Bfl-1 in melanoma

“…shown to arrest cell cycle in the G2 phase across a range of tumour types and it is the lack of progression across the G2/M transition which leads to apoptotic cell death 467,472,473 . The G2 arrest could be seen as the result of the formation of platinum adducts during the S phase of the cell cycle where DNA is replicated.…”

The FLT3 inhibitor tandutinib (formerly MLN518) has sequence-independent synergistic effects with cytarabine and daunorubicin

“…Furthermore, treatment with lestaurtinib with or following chemotherapy was found to be synergistic, whereas treatment with lestaurtinib followed by chemotherapy was generally antagonistic. 20 As combination of "cell cycle incompatible" drugs could result in antagonistic effects on killing of cancer cells, 20,35,[40][41][42] we examined cell cycle effects of the structurally unrelated FLT3 inhibitor tandutinib in combination with antineoplastic agents of the standard "3 + 7" regimen to define an optimal sequence for drug treatment.…”

“…Cell cycle experiments were assessed, as we have previously described, by flow cytometry after ethanol fixation and staining cells with propidium iodide (PI) (Fluka, Buchs, Switzerland). 35 Samples were pre-treated with RNAse to reduce RNA interference. We measured untreated, mono-treated and sequentially treated samples at 0 h, 24 h, 48 h, 72 h and 96 h-timepoints for dynamic analysis of cell cycle.…”

Mifepristone abrogates repopulation of ovarian cancer cells in between courses of cisplatin treatment

“…Another explanation for the repopulation of ovarian cancer cells post-CDDP that we observed in vitro is based on the fact that the culture was not synchonized and that cells are more prone to CDDP killing at particular times during the cell cycle. For instance, tumor testicular germ cells in vitro are more sensitive to the lethality induced by CDDP during the G2/M phase of the cell cycle than in other phases (39). Finally, there is a possibility that indeed CDDP is killing the population of differentiated cancer cells representing the bulk of the culture, but not the scarce tumor initiating cells with the capacity to regenerate the culture, and that appear to be resistant to most common DNA damaging agents (40).…”