piperidines and Carcinoma--Embryonal

Pluripotent mouse P19 embryonic carcinoma cells represent a convenient in vitro model for studying various aspects of cardiac differentiation. Here by using whole-cell patch-clamp recording we have identified the rapid delayed rectifier K+ current, I(Kr) in P19 cell induced to differentiate into cardiac phenotype by DMSO (1%). Cardiac differentiation was confirmed by the appearance of spontaneously beating cells, their morphological features, ultrastructural clusterization of mitochondria around contraction elements, expression of cardiac actin mRNAs and MLC2v, and by the presence of inward sodium and calcium currents. I(Kr) was isolated based on the sensitivity to the specific blocker, E-4031, which at concentration of 1 MM blocked more than 50% of the total outward K+ current. However, in contrast to I(Kr) in native cardiac myocytes and in heterologous systems expressing I(Kr)-carrying ERG1 potassium channel, E-4031-sensitive K+ current in cardiac-like P19 cells lacked characteristic inward rectification, suggesting specific regulation and/or subunit composition of endogenous ERG -based channel in these cells. Establishing the reason(s) for this phenomenon will advance the understanding of the mechanisms of I(Kr)-channel rectification. Cardiac-differentiated P19 cells might also be useful for studying pharmacological modulation of I(Kr), which is recognized target for cardiotoxic side effects of numerous drugs.

Topics: Actins; Animals; Calcium Channels; Carcinoma, Embryonal; Cell Differentiation; Cell Line, Tumor; Electrophoresis, Agar Gel; Ether-A-Go-Go Potassium Channels; Membranes; Mice; Microscopy, Electron; Mitochondria, Heart; Myocytes, Cardiac; Myosins; Patch-Clamp Techniques; Piperidines; Polymerase Chain Reaction; Potassium Channel Blockers; Pyridines; Sodium Channels

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.. 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.. 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.. 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.

Topics: Antineoplastic Agents; Apoptosis; Breast Neoplasms; Carcinoma, Embryonal; Cell Cycle; Cell Line, Tumor; Cisplatin; Dose-Response Relationship, Drug; Drug Synergism; Female; Flavonoids; Humans; Inhibitory Concentration 50; Ovarian Neoplasms; Paclitaxel; Piperidines; Uterine Cervical Neoplasms