The rate of etoposide-induced apoptosis is slower than that observed with receptor-mediated apoptosis. To attain a comparable level of apoptosis as seen with TRAIL, HeLa cells require 24h exposure to 50µM etoposide. This may be attributed to protein synthesis being required for etoposide-induced apoptosis as demonstrated by the ability of cycloheximide, a well-known protein synthesis inhibitor, to protect cells against etoposide-induced apoptosis (Chow et al., 1988). Unsurprisingly, etoposide-etoposide-induced apoptosis in non-synchronised cells was significantly inhibited in the presence of the pan-caspase-inhibitor, z-VAD-fmk, indicating that caspase activation is a required signal event for etoposide-induced apoptosis. However, Hoechst staining revealed little difference between the level of protection provided by caspase-2, -3 and -6. During etoposide-induced apoptosis caspase-2 inhibition prevents cytochrome c release, attenuates downstream events, such as pro-caspase-9 and -3 activation, phosphatidylserine exposure on the plasma
reflected in the level of protection provided by zVDVAD-fmk in our studies, but this appears not to be the case. The kinetics of caspase-3 and caspase-2 activation correlates with that of etoposide-induced nuclear apoptosis. As discussed previously in the TRAIL-induced apoptosis, caspase-2 and -6 inhibitors appear to inhibit DEVD-afc cleavage as effectively as caspase-3 and the pan-caspase inhibitor zVAD in etoposide-induced apoptosis in non-synchronised cells. Similarly, caspase-3 and -6 inhibitors appear to inhibit VDVAD-afc cleavage as effectively as caspase-2 and the pan-caspase inhibitor zVAD. Once again, we cannot exclude the possibility of cross-reactivity between the caspase inhibitors. A different caspase inhibitor profile was observed when DNA fragmentation was used as an apoptotic endpoint. The fact that the caspase inhibitor profile does not mirror the one observed by Hoechst staining suggests tat these two endpoints are executed by independent pathways. The cell death ELISA implicated caspase-6 as a major player in etoposide-induced DNA fragmentation. This finding appears to conflict with previous studies that suggest caspase-3 as the primary inactivator of DFF45/ICAD (DNA fragmentation factor-45/inhibitor of caspase-activated DNase) and therefore the primary activator of apoptotic DNA fragmentation (Wolf et al., 1999).
However, caspase and CAD independent DNA fragmentation also exists. Studies have demonstrated that another nuclease, endonuclease G (endoG), a mitochondrion-specific nuclease that translocates to the nucleus during apoptosis and is able to induce nucleosomal fragmentation of DNA independently of caspase and DFF/CAD(Li et al., 2001).
5.2.1 Cell synchronisation potentates etoposide toxicity
Since the expression and activity of topoisomerase II varies as a function of the proliferative status of the cell (Chow and Ross, 1987b), it was reasoned that a window might exist to rationally maximise cytotoxic drug efficacy. The high levels of topoisomerase II observed in late S compared to G1 phase is consistent with the enzyme being required mainly during the final stages of DNA replication (Tricoli et al., 1985).
Previous studies have shown that the fluctuation in enzyme activity correlates with the sensitivity to the DNA cleavage effects of topoisomerase-specific anti-tumour drugs (Paoletti, 1993). Sullivan et al. have demonstrated that the enzyme content of proliferating Chinese hamster ovary fibroblasts to be prominently higher than in quiescent cells and this corresponded well to an increased sensitivity of etoposide activity (Sullivan et al., 1987).
DISCUSSION
Further investigations have shown that the introduction of DNA strand breaks by etoposide is optimal in late S-phase (Smith et al., 1986).
On the basis of extensive literature demonstrating a positive relationship between topoisomerase II levelsand catatonic efficacy of drugs that target this enzyme, thelogical assumption was made that synchronisation of cells in S-phase would result in a homogeneous population with an elevated topoisomerase level which in turn, would lead to an increase in etoposide cytotoxicity. We achieved a relatively unperturbed synchronised population of HeLa by imposing a double thymidine block, which conferred amarked hypersensitivity to etoposide-mediated apoptosis. The increased sensitivity of the S-phase cells was similar to that observed by Freireich et al. in synchronised human lymphoma cells exposed to etoposide and in synchronised HeLa cells treated with m-AMSA (Freireich et al., 1984). Maximal potentiation of etoposide-induced DNA damage was observed upon 4h release from the second thymidine block, which corresponded with a peak of cells in S-phase as demonstrated by FACS analysis. The acceleration of etoposide-induced apoptosis in synchronised cells is evident. Following 2h etoposide exposure, synchronised cells exhibit 15% condensed nuclei, which is only attained after 6h treatment in non-synchronised HeLa cells. Cell synchronisation thus yielded a population of cells that would undergo apoptosis upon DNA damage almost simultaneously and in a short time period, thereby allowing a more direct comparison between etoposide and TRAIL-induced apoptosis concerning nuclear pore disassembly.
5.2.2 Etoposide induces cytochrome c release in synchronised HeLa cells
The release of cytochrome c from the mitochondria has been identified as one of the central events of apoptosis (Chandra et al., 2002). However, there is currently a level of uncertainty regarding the mechanism of cytochrome c release in response to specific DNA damaging agents. The mechanism of cytochrome c release is most commonly thought to be a caspase-independent process, with cytochrome c release preceding the activation of caspase-9 and caspase-3, (Karpinich et al., 2002). However there is evidence supporting caspase-dependent cytochrome c release from the mitochondria into the cytosol (Robertson et al., 2000a). Usually, caspase activity is not required upstream of cytochrome c, however
cytochrome c release during etoposide-induced apoptosis. Thus providing a link between etoposide-induced DNA damage and the engagement of the mitochondrial apoptotic pathway (Robertson et al., 2002)
In the present study, immunostaining revealed cytochrome c release in condensed cells exhibiting caspase-3 activation. This does not necessarily imply that cytochrome c release is caspase-dependent. It is more likely that cytochrome c release is upstream of caspase-3 activation, since the pan-caspase inhibitor, z-VAD-fmk could not completely abrogate cytochrome c release. Furthermore, cytochrome c was visible in caspase-3 negative cells and one may conclude that in these cells caspase-3 activation is inhibited despite cytochrome c release. The signals linking the release of cytochrome c in the absence of caspase activation are unclear. The cytosolic protein, Akt, a serine/threonine protein kinase has been implicated to play an important role in suppressing apoptosis at a post-mitochondrial stage, downstream of cytochrome c release and before activating caspase-9 (Zhou et al., 2000).
Robertson et al. have proposed a scheme whereby the concentration of etoposide determines cytochrome c release via two distinct pathways (Robertson et al., 2000a). On the one hand, low doses of the drug predominantly target the nucleus where damage to DNA triggers subsequent caspase-dependent eventsthat converge on the mitochondria and elicit cytochrome c release. In contrast, higher doses of etoposide are directly toxic to mitochondria,altering their ability to accumulate Ca2+, increasing their sensitivity to MPT, and stimulating the release of cytochrome c, an event that is not abolished by caspase inhibition (Robertson et al., 2000a). The concept of a caspase-dependent and independent pathway supports our findings. However, in our studies 50µM etoposide appears to induce cytochrome c release predominantly via the caspase-independent pathway. This difference may be attributed to the use of a Jurkat cell line in this study and perhaps the concentration threshold for determining which pathway predominates differs in HeLa 229 cells. The authors do conclude that two pathways are unlikely to be mutually exclusive i.e. it does not seem to bethe case that higher doses of etoposide only target mitochondria,whereas lower doses of the drug only damage DNA.
DISCUSSION
5.2.3 Bax translocation triggers cytochrome c release
We have observed endogenous Bax to be predominantly located in the cytosolic compartment, which is consistent with previous reports (Sarkar et al., 2003). However immuno-electron microscopy studies have described the presence of Bax at multiple subcellular localisations such as the nuclear outer membrane, endoplasmic reticulum membrane and mitochondrial membranes (Zong et al., 2003). Furthermore, Desagher et al.
have reported that in cultured cells, Bax was distributed in both the cytosol and mitochondria, whereas in freshly dissected tissues, Bax was found to have a predominantly cytosolic localisation and rarely detectable in the mitochondria (Desagher et al., 1999).
These apparent discrepancies may arise from differences in methodology used to determine Bax localisation or differences in cell line or culture conditions.
It has been widely reported that Bax redistributes from the cytosol specifically to the mitochondria during apoptosis induced by chemotherapeutic drugs (Zhang et al., 2000).
We have confirmed that this is the case when synchronised HeLa cells were apoptosis was induced with etoposide. Interestingly, a number of reports have provided circumstantial evidence that following a death signal Bax can also translocate to the cellular nucleus (Hoetelmans et al., 2000). The inhibition of signalling via the epidermal growth factor receptor in colorectal cancer cells resulted in increased expression of Bax, redistribution of the protein from the cytosol to the inner side of the nuclear membrane and apoptosis (Mandal et al., 1998).
Bax translocation from cytosol to mitochondria is believed to be a crucial step for triggering cytochrome c release from mitochondria (Degenhardt et al., 2002; Hardwick and Polster, 2002). Immunofluorecence studies confirmed that redistribution of Bax occurred in concert with cytochrome c release. Our findings are supported by work involving recombinant Bax, which has been shown to cause cytochrome c release from the mitochondria in vitro and intact cells (Degenhardt et al., 2002). On the contrary, it has also been reported that targeting of recombinant Bax to isolated mitochondria requires cytochrome c (Hardwick and Polster, 2002). In monitoring endogenous Bax localisation cytochrome c release was scarcely observed in the absence of Bax translocation, supporting a model in which Bax translocation precedes cytochrome c release.