Bcl-2 inhibits early apoptotic events and reveals post-mitotic multinucleation without affecting cell cycle arrest in human epithelial tumor cells exposed to etoposide |
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| Authors: | Mary Jane Elliott Kathleen M. Murphy Lolita Stribinskiene Velvizhi Ranganathan Eric Sturges Monica L. Farnsworth Richard B. Lock |
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| Affiliation: | (1) The Henry Vogt Cancer Research Institute, J. Graham Brown Cancer Center, Department of Medicine and Department of Biochemistry and Molecular Biology, University of Louisville, Louisville, KY 40202, USA, US;(2) Department of Microbiology and Immunology, University of Louisville, Louisville, KY 40202, USA, US;(3) Children's Cancer Research Institute, University of New South Wales, Sydney Children's Hospital, Randwick, Sydney 2031, Australia Tel.: +61-2-9382-1822; Fax: +61-2-9382-1850 e-mail: Richard.Lock@unsw.edu.au, AU |
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| Abstract: | Defective apoptotic mechanisms are considered to play a role in both the development of malignancy and resistance to chemotherapeutic drugs. The Bcl-2 family of proteins regulate the cellular commitment to survive or die when challenged with various apoptotic stimuli. Purpose: The purpose of this study was to identify the point at which Bcl-2 interrupts the apoptotic cascade initiated following exposure of human tumor cells to etoposide. Methods: A stable Bcl-2-expressing HeLa-transfected clonal cell line, along with its control-vector-transfected counterpart, were utilized in this study. Following etoposide exposure, cells were examined for cell cycle arrest, formation of hyperdiploid cells, apoptotic DNA degradation, loss of plasma membrane integrity, levels of expression of members of the Bcl-2 protein family, caspase activation, degradation of poly(ADP-ribose) polymerase and movement of Bax from cytosol to cellular membrane fractions. Results: Caspase activation, poly(ADP-ribose) polymerase degradation and Bax membrane insertion were initiated rapidly following etoposide removal, concomitantly with cell cycle arrest. Whereas Bcl-2 had no effect on etoposide-induced cell arrest, it interrupted all aspects of apoptosis, including activation of caspases, poly(ADP-ribose) polymerase degradation, DNA fragmentation and loss of plasma membrane integrity. Surprisingly, Bcl-2 also blocked Bax membrane insertion. In addition, Bcl-2 also prevented the increase in cellular levels of Bak, Bax and Bcl-xL, along with degradation of actin and Bax. However, inhibition of etoposide-induced apoptosis by Bcl-2 resulted in the accumulation of giant, multinucleated cells that eventually lost the ability to exclude trypan blue without apoptotic morphology or DNA degradation. Conclusions: These results indicate that biochemical apoptotic processes are initiated concomitant with etoposide-induced cell cycle arrest and are interrupted by Bcl-2 overexpression. However, the aberrant mitotic events induced by etoposide are sufficient to kill these cells even in the absence of apoptosis. Received: 1 September 1998 / Accepted: 3 November 1998 |
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| Keywords: | Apoptosis Bax Bcl-2 Cell cycle check-points Etoposide |
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