Time-dependent changes in factors involved in the apoptotic process in human ovarian cancer cells as a response to cisplatin.
OBJECTIVE
Apoptosis is believed to be a major mechanism of cisplatin-induced cell death. We investigated the kinetics of apoptosis in four human ovarian cancer cell lines treated with cisplatin to obtain insight into the role and the behavior of a variety of factors involved in this process.


METHODS
The cell lines A2780, H134, and IGROV-1 (all wild-type p53) and OVCAR-3 (mutant p53) were exposed to cisplatin for 1 h and the antiproliferative effects were measured after 96 h. At various time points up to 96 h after the 1-h exposure to the individual 90% growth-inhibiting cisplatin concentrations, FACS analysis and May-Grünwald Giemsa staining were carried out to determine the extent of apoptosis. At the same time points protein expression levels of p53, p21/WAF1, Bax, and Bcl-2 and the activity of caspase-3 were measured. FACS analysis was also carried out to determine changes in cell cycle distribution as a response to cisplatin.


RESULTS
The four cell lines differed in sensitivity to cisplatin. A2780 was the most sensitive and IGROV-1 was the least sensitive. In contrast, IGROV-1 cells showed the highest percentage of apoptosis (30-40%), while A2780 had the lowest percentage (6-14%) (r = 0.99). The occurrence of apoptosis was not dependent on functional p53. Of interest, caspase-3 activity was in line with the percentage of apoptosis and preceded DNA fragmentation and the visualization of condensed nuclei. Wild-type p53 cells accumulated in the S phase, while OVCAR-3 arrested in the G2/M phase. The protein expression levels of p53, p21/WAF1, Bax, and Bcl-2 varied in time, but were not related to the apoptotic behavior of the cells. Upregulation of p53 was already evident before activation of caspase-3.


CONCLUSIONS
Time-dependent changes in the various factors involved in the apoptotic process induced by equitoxic doses of cisplatin vary strongly among the cell lines. Caspase-3 activation plays an important role in cisplatin-induced apoptosis and this precedes morphological changes. The ability of cells to enter apoptosis, however, does not seem to predict sensitivity to cisplatin.
Kolfschoten
G M
GM
Department of Medical Oncology, Vrije Universiteit Medical Centre, Amsterdam, the Netherlands.
Hulscher
T M
TM
Schrier
S M
SM
van Houten
V M M
VM
Pinedo
H M
HM
Boven
E
E
D000970:Antineoplastic Agents / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects* / Q000502:physiology; D053148:Caspase 3; D020169:Caspases / Q000096:biosynthesis / Q000502:physiology; D002453:Cell Cycle / Q000187:drug effects / Q000502:physiology; D002945:Cisplatin / Q000494:pharmacology*; D050759:Cyclin-Dependent Kinase Inhibitor p21; D016213:Cyclins / Q000096:biosynthesis / Q000502:physiology; D004789:Enzyme Activation; D005260:Female; D006801:Humans; D020128:Inhibitory Concentration 50; D010051:Ovarian Neoplasms / Q000188:drug therapy / Q000378:metabolism / Q000473:pathology*; D011518:Proto-Oncogene Proteins / Q000096:biosynthesis / Q000502:physiology; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000096:biosynthesis / Q000502:physiology; D014407:Tumor Cells, Cultured; D016159:Tumor Suppressor Protein p53 / Q000096:biosynthesis / Q000502:physiology; D051028:bcl-2-Associated X Protein
D000970:Antineoplastic Agents; C494826:BAX protein, human; C493956:CDKN1A protein, human; D050759:Cyclin-Dependent Kinase Inhibitor p21; D016213:Cyclins; D011518:Proto-Oncogene Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; D016159:Tumor Suppressor Protein p53; D051028:bcl-2-Associated X Protein; C505407:CASP3 protein, human; D053148:Caspase 3; D020169:Caspases; D002945:Cisplatin