Bcl-2 inhibits early apoptotic events and reveals post-mitotic multinucleation without affecting cell cycle arrest in human epithelial tumor cells exposed to etoposide

Defective apoptotic mechanisms are considered to play a role in both the de velopment 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 st udy was to identify the point at which Bcl-2 interrupts the apoptotic casca de initiated following exposure of human tumor cells to etoposide. Methods: A stable Bcl-2-expressing HeLa-transfected clonal cell line, along with it s control-vector-transfected counterpart, were utilized in this study. Foll owing etoposide exposure, cells were examined for cell cycle arrest, format ion of hyperdiploid cells, apoptotic DNA degradation, loss of plasma membra ne integrity, levels of expression of members of the Bcl-2 protein family, caspase activation, degradation of poly(ADP-ribose) polymerase and movement of Bar from cytosol to cellular membrane fractions. Results:Caspase activa tion, poly(ADP-ribose) polymerase degradation and Bar membrane insertion we re initiated rapidly following etoposide removal, concomitantly with cell c ycle 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 plas ma membrane integrity. Surprisingly, Bel-2 also blocked Bar membrane insert ion. In addition, Bcl-2 also prevented the increase in cellular levels, of Bak, Bar and Bcl-x(L), along with degradation of actin and Bar. However, in hibition of etoposide-induced apoptosis by Bcl-2 resulted in the accumulati on of giant, multinucleated cells that eventually lost the ability to exclu de 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 b y Bcl-2 overexpression. However, the aberrant mitotic events induced by eto poside are sufficient to kill these cells even in the absence of apoptosis.