title: Induction of apoptosis by flavopiridol unrelated to cell cycle arrest in germ cell tumour derived cell lines.
abstract: BACKGROUND
  Germ cell tumours (GCTs) are highly sensitive to cisplatin-based chemotherapy. The inability to arrest the cell cycle at the G1/S-check-point due to a lack of retinoblastoma gene product RB has been suggested as one potential explanation for this feature. Flavopiridol (FP), an inhibitor of cyclin dependent kinases, causes cell cycle arrest or apoptosis depending on the relation of the transcription factor E2F1 and RB.
  
  
  METHODS
  The effect of FP was evaluated in GCT-derived cell lines NT2, 2102 EP and NCCIT in comparison to cell lines derived from ovarian cancer (SKOV), breast cancer (MCF7), and cervical cancer (HeLa) using the MTT-assay. Cell cycle progression and induction of apoptosis were assessed by flow cytometry and immunoblot analysis of PARP-cleavage.
  
  
  RESULTS
  FP did not affect cell cycle progression and proliferation of GCT cell lines at sublethal doses. At higher concentrations, cell death occurred independent of cell cycle progression. The IC50 was approximately fivefold lower for the three GCT cell lines (60/60/70 nM) than for the other tumour cell lines tested (350/280/300 nM). Lethal doses in vitro were markedly lower than plasma concentrations of FP achieved in clinical studies. In vitro sensitivity to FP did not correlate with that to cisplatin. The cell lines NTera2 and NCCIT showed comparable responses to FP despite differing in their IC50 to cisplatin by factor 4. Flow cytometry and immunoblot for PARP indicated apoptotic cell death induced by FP. Synergism between either cisplatin or paclitaxel and FP was not observed. However, at low concentrations, cytotoxicity of FP and cisplatin appeared to be additive.
  
  
  CONCLUSIONS
  These prelinical investigations suggest a significant antitumour activity of FP in GCT. GCT derived cell lines were far more responsive to FP than cell lines derived from other solid tumours. In contrast to other models, FP did not induce cell cycle arrest in the GCT-derived cell lines tested, possibly due to the known lack of RB-expression in GCTs. However, apoptosis was induced unrelated to cell cycle progression already at low concentrations. No cross resistance between FP and cisplatin was observed. A clinical trial evaluating the activity of FP in patients with cisplatin-refractory GCTs appears to be warranted.
authors.affiliation: Department of Oncology, Hematology, Immunology, and Rheumatology, University of Tübingen Medical Center, Otfried-Mueller-Str. 10, Tübingen, Germany.
authors.forename: Frank
authors.initials: F
authors.lastname: Mayer
authors.forename: Sandra
authors.initials: S
authors.lastname: Mueller
authors.forename: Elke
authors.initials: E
authors.lastname: Malenke
authors.forename: M
authors.initials: M
authors.lastname: Kuczyk
authors.forename: Jörg T
authors.initials: JT
authors.lastname: Hartmann
authors.forename: Carsten
authors.initials: C
authors.lastname: Bokemeyer
mesh_terms: D000970:Antineoplastic Agents / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects*; D001943:Breast Neoplasms / Q000473:pathology; D018236:Carcinoma, Embryonal / Q000473:pathology*; D002453:Cell Cycle / Q000187:drug effects*; D045744:Cell Line, Tumor; D002945:Cisplatin / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D004357:Drug Synergism; D005260:Female; D005419:Flavonoids / Q000494:pharmacology*; D006801:Humans; D020128:Inhibitory Concentration 50; D010051:Ovarian Neoplasms / Q000473:pathology; D017239:Paclitaxel / Q000494:pharmacology; D010880:Piperidines / Q000494:pharmacology*; D002583:Uterine Cervical Neoplasms / Q000473:pathology
chemical_list: D000970:Antineoplastic Agents; D005419:Flavonoids; D010880:Piperidines; C077990:alvocidib; D017239:Paclitaxel; D002945:Cisplatin