Despite the considerable amount of information on apoptosis in tumour cell lines acquired from cytometry-based studies, little attention seems to have been paid to the time course of the main events that characterise the apoptotic process, and in particular those induced by taxanes.
Earlier studies established that phosphatidylserine exposure to the outer membrane leaflet occurs simultaneously with chromatin condensation  and precedes DNA fragmentation . Other membrane changes, as shown by an increase in Merocyanin 540 binding of apoptotic cells [18–20], may not be independent of the phosphatidylserine contribution to the membrane lipid composition . On the other hand, AnnV staining itself may be lacking in certain apoptotic cell populations . These findings underline the need to evaluate multiple parameters when assessing cell death. A number of studies have addressed the issue of mitochondrial involvement in the apoptotic process by evaluating Δψm decrease with different dyes [22–24] inan attempt to establish a temporal – and possibly – causal relationship within the events that characterise apoptosis. It appears that the bcl-2 content of the cells may be related to the timing of mitochondrial involvement . This consideration is particularly important in taxane-induced apoptosis, where bcl-2 is involved in the cell death induction mechanisms as well as in drug resistance [26–28].
Taxanes and their derivatives inhibit microtubule depolymerisation, thus preventing cell cycle completion  and thus causing apoptotic cell death. A similar effect is also caused by drugs that inhibit microtubule polymerisation, such as vinblastine and vincristine. Apoptosis triggered by agents that disrupt microtubule organisation is associated with bcl-2 phosphorylation and is independent of p53 involvement, at variance with apoptosis due to DNA-damaging agents . Despite these differences, both microtubule disruption and DNA damage activate the intrinsic pathway of cell death, causing the release of mitochondrial components and the formation of apoptosomes. In both cases, the triggering mechanism is believed to involve changes in the amount and/or activity of anti- and pro-apoptotic members of the bcl-2 family.
Conversely, it has been shown that administration of paclitaxel – the prototype for taxane compounds – causes an almost immediate increase in cytosolic Ca2+, associated with a rapid decline in mitochondrial membrane potential, presumably due to the opening of the permeability transition pores (mPTPs) [11, 31]. The rapidity of this event apparently rules out the participation of mechanisms controlling microtubule assembly status, thus suggesting a pathway of death induction different from that previously reported . However, it is recognised that loss of Δψm and variations in [Ca2+]i may be transient and do not necessarily lead to apoptosis. In particular, mPTPs have two conformations, one characterised by low- and the other by high-conductance state . The opening of the former is reversible, allows the release of small molecules only – such as calcium ions – and may be involved in the rapid [Ca2+]i changes described by other authors . In contrast, late changes in Δψm, such as those observed by us 16 hours after drug treatment, are likely to involve the high-conductance mPTP, which is responsible for the release of apoptosis-inducing compounds. The delayed change in Δψm is not apparently affected by prior exposure to CsA (table 3). However, it is obvious that cell cycle is already deeply affected as early as 6 hours after the end of drug treatment (Fig. 2). Thus, a more realistic view puts the mitochondrial involvement in apoptosis downstream of paclitaxel-induced G2/M arrest. Within this cell-cycle phase, microtubule impairment may activate mTOR kinase, thus affecting bcl-2 phosphorylation and its subsequent proteasome-dependent degradation . The data on the action/activity of CsA presented in the present study are only preliminary and the role played by Ca2+ variation in this process will be the object of future investigation.
Whilst the time course of early events has yet to be defined, its eventual clarification will provide new insight into the mechanism whereby taxanes induce cell death. Moreover, other well-studied events that belong to the execution phase of apoptosis have not yet been placed in a precise time setting. These include phosphatidylserine exposure to the outer cell surface, DNA cleavage, and loss of membrane integrity. Changes in surface glycosylation are less well understood and have been included here because they currently being studied by the authors (Marini et al., manuscript in preparation). Our results suggest that these changes are not peculiar to a given cell type and potentially represent a new marker of apoptosis. They would seem to occur about the same time as PS exposure to the outer surface and may be related to it.
As described in the present work, apoptotic events in the HT1376 bladder cancer cell line treated with DOC would appear to occur in the following order: 1) cell cycle arrest; 2) loss of mitochondrial membrane potential; 3) cell shrinkage and decrease in granularity; 4), DNA fragmentation (hypodyploid peak and electrophoretic laddering); 5) and 6) change in syalilation of surface proteins and loss of surface phospholipid asymmetry; 7) loss of membrane integrity (cells become PI+); 8) further DNA fragmentation (TUNEL). The above outlined sequence of events takes into account intrinsic limitations in cytometric methods, which include a lesser degree of sensitivity in comparison with biochemical determinations and the variability in drug response within an asynchronous cell population. In fact, it must be stressed that all the cells do not die simultaneously after drug treatment because death is dependent on factors such as the cell cycle status and the individual energy level. Moreover, in most artificial systems, e.g. culture flasks, cells dying by apoptosis are not phagocyted by competent cells and eventually lose their membrane potential and undergo secondary necrosis. Thus, within an asynchronously growing cell population, cycling cells, cycle-arrested cells, cells undergoing apoptosis and cells already undergoing secondary necrosis can all be found and will be sending out conflicting signals that require interpretation.
Data on DOC-treated U937 myelomonocytic cell lines were reported in the present work for comparison purposes and were not as complete as those obtained on HT 1376 bladder cancer cell line. Nevertheless, they suggest that Δψm is an early event in both cell types, that bear though different they may be, and that MAA marks the same cell subset as AnnV.
In permeabilised cells, DNA fragmentation results in decreased PI staining (i.e. cells appear to have a hypodiploid DNA content) . Accordingly, this event appears to occur between 24 and 48 hours after drug administration. However, the onset of DNA fragmentation may occur earlier, as cells with duplicated DNA join the sub-G0/1 peak after passing through the S and G0/1 peaks. On the other hand, it is possible that, over time, the progressive increase in DNA fragmentation causes a marked loss in PI stainability, causing the hypodiploid peak to be underestimated, as suggested by comparing data with results obtained from unpermeabilised cells and from TUNEL staining, another index of DNA fragmentation. This technique, where 3' OH termini – resulting from DNA cleavage – are labeled with FITC-conjugated nucleotides in a reaction utilising exogenous terminal deoxynucleotide transferase (TdT), is apparently less sensitive than a decrease in PI stainability, possibly because it requires more extensive DNA cleavage. However, for longer culture times, TUNEL staining appears to give a better estimate of the percentage of apoptotic cells with respect to the evaluation of the hypodiploid peak. In fact, 96 hours after DOC treatment, the percentage of TUNEL+ cells was much higher than that of cells in the sub-G0/1 peak, and approximated that of AnnV+ cells.
The characteristic ladder revealed by gel electrophoresis of DNA from DOC-treated cells demonstrates that chromatin fragmentation did not occur randomly, but rather at internucleosomal sites. Neither high molecular weight DNA cleavage (as occurs during the initial stages of apoptosis), nor aspecific DNA fragmentation (following necrotic cell death), produce such a specific pattern of electrophoretic migration. The same applies to the formation of the hypodiploid peak .
Cell surface modifications were observed 24 hours after DOC treatment and over time extended to other cells (table 1). Due to its strong affinity to negatively charged phospholipids, AnnV binds phosphatidylserine as it becomes exposed to the outer cell surface, a common feature of cells undergoing apoptosis. Variations in cell surface carbohydrate composition can be identified by changes in lectin-mediated recognition. Apoptotic cells may change the oligosaccharide composition of their glycocalix, thus displaying a differential binding to some lectins. In particular, Maakia Amurensis lectin (MAA), which preferentially recognises galactose residues bound to α 2,3 sialylic acid , was found to bind to apoptotic cells rather than to their "healthy" counterpart (Marini et al, manuscript in preparation). The percentage of cells showing variations in the glycosylation of surface molecules initially increased more sharply than that of cells displaying phosphatidylserine on the outer membrane leaflet. However, over time the percentage of MAA+ cells did not reach that of AnnV+ cells. Studies are ongoing to further the understanding of these variations in surface syalilation and of their relevance in apoptosis. As already pointed out, the cell population appeared to acquire both MAA and AnnV markers before losing its membrane integrity (i.e. before becoming permeable to PI). In addition to such information, the percentages of apoptotic PI- and PI+ cells observed shows that the increase in TUNEL positivity (i.e. progression in DNA degradation) also occurred in cells that had lost membrane integrity.
Ninety-six hours after drug treatment, the near totality of cells displayed a decrease in transmembrane mitochondrial potential Δψm that accompanied the efflux of pro-apoptotic molecules from the mitochondrion, as evaluated by the cationic fluorochrome JC-1 (table 1). This suggests that almost all of the DOC-treated cells eventually undergo apoptosis. Moreover, it supports the view hypothesis that mitochondrial events occur very early during cell culture and pave the way for both DNA cleavage and surface modifications. Furthermore, cytoskeleton modifications that cause cell shrinkage and loss of granularity (Fig. 4) may be subsequent to a loss of Δψm, as suggested elsewhere .
In the present paper we compared cytofluorimetric with non cytofluorimetric methods of evaluation of cell death. The same conclusions about the cytotoxic and cytostatic effects of the drug were reached using the SRB assay, the dye-exclusion test and the cytofluorimetric evaluation of cell cycle. Apoptosis-induced cell surface and chromatin modifications can be assessed by both morphological evaluation and cytometry. However, cytometry appears to be better suited for quantification purposes due to its simplicity of use and to the wide range of applications that are made possible by technical improvements.