- Original Articles
- Open Access
Taxol-Induced Mitotic Block Triggers Rapid Onset of a p53-Independent Apoptotic Pathway
© Molecular Medicine 1995
- Published: 1 July 1995
At therapeutic concentrations, the antineoplastic agent taxol selectively perturbs mitotic spindle microtubules. Taxol has recently been shown to induce apoptosis, similar to the mechanism of cell death induced by other antineoplastic agents. However, taxol has shown efficacy against drug-refractory cancers, raising the possibility that this pharmacological agent may trigger an alternative apoptotic pathway.
Materials and Methods
The kinetics and IC50 of mitotic (M) block, aberrant mitosis, and cytotoxicity following taxol treatment were analyzed in human cell lines as well as normal mouse embryo fibroblasts (MEFs) and MEFs derived from p53-null mice. Apoptosis was followed by DNA gel electrophoresis and by in situ DNA end-labeling (TUNEL).
Taxol induced two forms of cell cycle arrest: either directly in early M at prophase or, for those cells progressing through aberrant mitosis, arrest in G1 as multimininucleated cells. TUNEL labeling revealed that DNA nicking occurred within 30 min of the arrest in prophase. In contrast, G1-arrested, multimininucleated cells became TUNEL positive only after several days. In the subset of cells that became blocked directly in prophase, both wt p53-expressing and p53-null MEFs responded similarly to taxol, showing rapid onset of DNA nicking and apoptosis. However, p53-null MEFs progressing through aberrant mitosis failed to arrest in the subsequent G1 phase or to become TUNEL positive, and remained viable.
Taxol induces two forms of cell cycle arrest, which in turn induce two independent apoptotic pathways. Arrest in prophase induces rapid onset of a p53-independent pathway, whereas G1-block and the resulting slow (3–5 days) apoptotic pathway are p53 dependent.
Taxol is a complex plant alkaloid with a unique taxane ring structure which appears to target microtubules (MTs) specifically both in vitro and in vivo (1–6). Taxol binds to the polymeric microtubule form of tubulin in a 1:1 stoichiometry with αβ-tubulin heterodimer subunits (2,4,7). Binding markedly shifts the dynamic instability equilibrium of MT polymers, stabilizing against dissociation and hence augmenting polymerization. Bound taxol overrides the need for GTP hydrolysis in tubulin heterodimer polymerization, and consequently it also serves to lower the critical concentration for tubulin polymerization (2,7); in vivo this is manifested by the ability of taxol to override the centrosomal MT nucleating activity in interphase cells, resulting in the appearance of non-centrosomally linked MT bundles throughout the cytoplasm (1,5,6,8). Currently, there has been considerable interest in taxol as a cancer therapeutic agent, in particular because it has shown activity against leukemias, late stage ovarian, and metastatic breast cancers refractory to standard chemotherapeutic treatments (9–12). Phase I and II trials have also shown some efficacy against advanced small cell lung cancer (13), melanoma (14,15) and head and neck cancers (16).
Microtubules constitute a major class of structural filaments in the eukaryotic cell and play a key role in a wide repertoire of cellular processes: endocytosis, vesicular movement, cell morphology and motility, and mitosis to name but a few (17). Somewhat surprisingly, however, taxol concentrations within the therapeutic range are not necessarily sufficient to cause gross rearrangements of interphase MT arrays and leave a functional MT scaffold intact. Therefore, it is not immediately apparent why taxol should be cytotoxic rather than merely cytostatic. In cultured human cell lines, taxol has been shown to induce a block in the cell cycle at the G2/M transition (1,18–23). At the G2/M transition, cellular MTs undergo a dramatic reorganization associated with a sudden change in the centrosomal MT-nucleating activity (5,6,23,24). Interphase MTs dissociate (even in the presence of taxol) as the centrosomes concurrently elaborate the mitotic spindle MTs (5,6,23,24). Interestingly, this sudden change in centrosomal activity correlates with association of p34cdc2/cyclin B complex with the centrosome (23,25). Taxol induces aberrant multipolar spindle formation that causes mitotic arrest (5,6,26). Recent studies with human lines have provided strong evidence that it is this arrest in M that correlates with cytotoxicity (18,20–22,27,28). For HeLa cells, the EC50 for arrest at the G2/M transition is similar for the EC50 for taxol-induced cytotoxicity and 1 to 2 orders of magnitude lower than the doses required to induce gross changes in interphase MT arrays (18). Rodent cells are also sensitive to taxol in ranges that perturb mitosis but appear to be more tolerant of arrest in mitosis; they eventually assume a multimininucleated phenotype resulting from aberrant mitosis and lack of cytokinesis. Doses effecting these changes are also cytotoxic (5,6,19). Species differences between human and rodent lines have been attributed to differences in p34cdc2 regulation (19). Taken together, these studies predict that taxol should be therapeutically active only against cells that are rapidly cycling. In balance, this appears to be the case, and the side effects of neutropoenia and leukopoenia as well as gastrointestinal-related side effects have been shown to be consistent with the hypothesis that taxol effects are due to mitotic arrest (29,30). However, exceptions exist where tumor regression is found despite the lack of actively cycling tumor cells and studies with leukemic lines have indicated that sensitivity to taxol correlates with the induction of MT bundles rather than multipolar aster formation (8,31). Therefore, the nature of taxol’s therapeutic activity remains to be fully elucidated.
Cell death can be induced by either necrosis or an active process known as apoptosis (32–36). The latter appears to be closely linked to the programmed cell death that occurs in development (32–35). Typically, high levels of insult induce necrosis, whereas lower levels induce apoptosis (33,35,36). In several cases of cellular differentiation, growth factors serve not only as proliferative and differentiating signals but also serve to rescue precursor proliferating cells from apoptosis (33–35). Thus certain cell types, most dramatically cells of the hematopoetic lineages, are particularly poised for apoptosis (low threshold for apoptosis defined in Refs. 33 and 35). It has now been well established that DNA-damaging cancer therapeutics and γ-irradiation exert their cytotoxic effects by inducing the programmed apoptotic pathway following arrest in G1 or G2 as opposed to necrosis (36–42). The tumor suppressor, p53, has been shown to play a key role in this process. The process of DNA nicking in and of itself induces elevated p53 levels (43,44) which in turn induce G1 arrest by up-regulation of the cdk inhibitor WAF1/Cip1/p21 (45–48) to prevent progression past the G1/S checkpoint of cells with damaged DNA. Extensive DNA damage leads to prolonged G1 arrest and apoptosis (43). Similarly, commonly used antineoplastic agents that target DNA, which induce arrest in G1 or G2, trigger the same p53 response (42–44,49–55). Interestingly, many advanced stage tumor types which have poor prognosis with established antineoplastic therapies express a high percentage of p53 mutations (55–59). For many tumor types, a late stage event in the progression of the cancer is the clonal expansion of cells expressing mutant p53 alleles (55–59). Mutation of the normal cellular p53 gene has been shown to be sufficient to induce cellular immortalization (55,59,60) and can enhance tumorigenicity (61–64). p53 normally functions as a homo-oligomer; hence, a single mutant p53 allele can lead to a dominant negative phenotype (63,65). The discovery that Li Fraumeni patients, genetically predisposed to an abnormally high incidence of cancer, fail to mount a normal p53-mediated response, together with studies indicating that transgenic mice carrying germ line knock-out of p53 alleles are more prone to develop spontaneous cancers and fail to mount an appropriate apoptotic response to γ-irradiation or DNA-damaging antineoplastic agents, provides strong support for the hypothesis that p53 plays an important role in the apoptotic pathways underlying elimination of aberrant tumor cells (42–44,49,50,52–55,61,66,67). Alternative non-p53-dependent apoptotic pathways also exist. These operate in normal development; for example apoptosis resulting from growth-factor deprivation appears to occur via a p53-independent pathway (33,35,52,54,68). Since taxol induces predominantly arrest in early M rather than G1 or G2 arrest in human cells, it is of interest to know whether the apoptotic pathway triggered by taxol (69–71) is dependent on p53.
Here we describe detailed comparative analyses of taxol effects on several human carcinoma lines as well as a normal human line. Kinetic analysis reveals that the majority of human cells become blocked at the G2/M transition in prophase, with only a small percentage progressing through a protracted aberrant mitosis, manifested by a multimininucleated phenotype, to become arrested in the subsequent G1 stage. The lag between mitotic block and cell death is 20–30 hr, with similar EC50 values for both processes. G1-arrested multimininucleated cells are also destined to die but with much slower kinetics (several days). We further demonstrate that both forms of taxol-induced cell cycle arrest lead to apoptosis. However, apoptosis triggered by arrest in M is initiated very rapidly, within 30 min of arrest at prophase, and appears to operate by a p53-independent pathway. In contrast, both the G1 arrest and subsequent apoptotic death of cells progressing through an aberrant mitosis are p53 dependent.
Hela, Hs578T, Hs578Bst, MCF7, and Rat1 cells were all obtained from ATCC and each grown in the medium specified by ATCC at 37°C in a humid atmosphere containing 5% CO2. wt p53 Mouse embryo fibroblasts (MEFs) were isolated by trypsinizing the muscle mass of 13-day mouse embryoes, filtering through 70-µm filters and plating at 0.3 × 106 cells per cm2 in DMEM (Gibco, Gaithersburg, MD, U.S.A.) containing 10% FBS (ICN) and 1 × penicillin/streptomycin (Gibco). After 1 h nonadherent cells were removed. MEFs were passaged twice before being used for experiments. MEFs lacking p53 (p53−/− MEFs) were obtained from T. Jacks (MTT). p53 status was verified by Western blot analyses. For comparative nuclear and viability/growth analyses, cells were seeded both in 48-well plates at 2.5 × 104 cells per well (for viability and cell counting) and onto #1 glass coverslips in 6-well plates at 5 × 105 cells per well (for mitotic scoring). Twenty-four hours later, the medium was replaced with medium containing taxol at the specified concentrations. Taxol was always added directly from a DMSO stock to prewarmed medium immediately prior to treating the cells; final DMSO concentration was always less than 0.1 %. DMSO up to 1 % had little effect on cell growth. At specified times, nonadherent cells were collected and the adherent cells trypsinized. After pelleting briefly at 2,000 rpm for 2 min in an Eppendorf centrifuge, the cell pellet was resuspended in a final volume of 50–100 µl 2% Trypan blue in PBS. Concurrently, coverslips were fixed in Omnifix for 10 min then stained with 2 µg/ml Hoechst 33342 (Boehringer-Mannheim, Indianapolis, IL) in PBS. To assay cells in suspension, 0.5 ml medium was removed after very gentle swirling, cells pelleted and stained in 2 µg/ml Hoechst in 30% ethanol. Stained cells were viewed under epifluorescence with a Zeiss Axiophot microscope. Two sets of 250 cells, located by random walk of the stage control, were scored as prophase, mitotic, multimininucleated or normal interphase for each time point. The ratio of attached to nonadherent cells derived from the total cell counts was used to convert scoring of Hoechst-stained adherent and nonadherent cells to overall percentages in the total population.
To obtain cells synchronized in the cell cycle, mitotic cells were collected by selective shake-off from 70 to 80% confluent (higher densities helped minimize detachment of interphase cells) HeLa cells with two successive 7-min incubations at 37°C in Ca2+ and Mg2+-free Hanks’ buffered salt solution (CMF-HBSS; 37°C). Harvested cells were cooled to 15°C to arrest cells in mitosis without depolymerizing the MTs, and collected by centrifugation at 15°C for 5 min at 1000 × g. A small aliquot of cells was fixed and Hoechst-stained to assess percent mitotic cells and the remainder plated onto coverslips or incubated as required. Only preparations exceeding 50% mitotic cells were used for experiments.
End Labeling of Cellular DNA and Agarose Gel Electrophoresis
Cellular DNA was isolated from cells at different times following taxol treatment by proteinase K and RNAaseA digestion of cell lysates followed by phenol chloroform extraction and ethanol precipitation following standard protocols. DNA was end labeled with 32P-γ ATP using terminal transferase (Boehringer Mannheim), electrophoresed on a 1.4% agarose gel and transferred to nitrocellulose. The dried blot was exposed to Kodak X-OMAT film for 15–30 hr at −80°C.
TUNEL Labeling and Immunofluorescence
For tubulin immunofluorescence (IF) cells were fixed in −20°C methanol and processed for IF using anti-β tubulin mAbs (Amersham, Arlington Heights, IL). For labeling DNA ends in situ, the TUNEL method described by Gavrieli et al. (72) was modified as follows. Cells were fixed for 5 min in 2% formaldehyde in TBS (10 mM Tris HCl (pH 8.0), 100 mM NaCl, 1 mM MgCl2, 1 mM EDTA) and permeabilized in 0.5% Triton X-100 in TBS for 15 min. After successive washes in TBS and TTD buffer (100 mM Na cacodylate (pH 6.6), 5 mM CoCl2, 0.26 mg/ml BSA) cells were end-labeled with terminal transferase using digitonin-coupled dUTP (Boehringer Mannheim) followed by counter labeling with FITC-conjugated mouse anti-digitonin mAbs as described (Boehringer Mannheim). For this application of terminal transferase activity, we found considerable variation in enzyme batches. To ensure consistent results the following control was included. Fixed cells were treated with 50 µl/ml micrococcal nuclease (Boehringer Mannheim) in 10 mM Tris HCl (pH 8.5), 1 mM MgCl2, 1 mM CaCl2 for 20 min and then processed for TUNEL labeling as described above. Only enzyme batches that yielded clean nuclear staining following this treatment were used for these experiments. In addition, etoposide treated cells were also used as controls for a positive reaction. To assess the kinetics of DNA nicking following taxol-induced arrest at the G2/M transition, mitotic cells harvested by selective shake-off were plated onto coverslips and incubated for 10 hr at 37°C. One hundred nanomolar taxol was then added and after an additional 3–4 hr, coverslips were collected at 30-min intervals, fixed, processed for TUNEL staining, and counterstained with Hoechst 33342. Nonadherent cells were also processed. Two sets of 250 cells per coverslip were scored. Control untreated cells typically exhibited a peak of mitosis between 16–17 hr after the shake-off.
Kinetics of Mitotic Block and Cytotoxicity Following Taxol Treatment
Comparison of EC 50 values for mitotic arrest and cytotoxicity induced by taxol in various human cell lines
Mitotic Arresta (nM)
Aberrant Mitosisa (nM)
8.4 ± 1.5
8.4 ± 1.5
25.6 ± 5.6
16.6 ± 7.6
10.0 ± 5.3
14.8 ± 4.9
18.5 ± 6.5
12.5 ± 0.5
6.7 ± 1.7
6.7 ± 0.5
47.0 ± 7.2
Anti-tubulin IF revealed that 2–4 hr of exposure to taxol above 10 nM induced abnormal multipolar spindles in the prophase-blocked cells (Fig. 3 C and D) compared with the normal bipolar spindle arrays of untreated cells (Fig. 3B). In contrast, gross rearrangements of MT arrays in interphase cells required concentrations in the 1–10 µM range (5,6,73). As shown in Table 1, the EC50 values for taxol-induced cytotoxicity at 72 hr correlated with the EC50 for arrest in early M (7–18.5 nM) and were two orders of magnitude lower than the 1–10 µM concentrations required for gross perturbations of interphase MT arrays (73). The EC50 values for M arrest and cytotoxicity were similar for all human lines examined. No difference was noted between the tumor-derived lines and the Hs578Bst line derived from normal breast tissue.
Taxol-induced Cytotoxicity Occurs by an Apoptotic Pathway
Bulk DNA analysis does not reveal whether apoptosis is selectively occuring in those cells that are blocked in M at prophase. To address this issue we performed in situ digitonin-dUTP end-labeling (TUNEL, 72). Since enzyme batches were highly variable in this assay, and the procedure was found to be very sensitive to permeabilization and fixation conditions, we routinely used as a control, normal cells that had been briefly treated for 20 min with 50 µl/ml miccrococcal nuclease after fixation before being processed for TUNEL labeling (Fig. 5 I and J). Only batches of terminal transferase that gave uniformly bright-staining nuclei after in situ nuclease digestion were used. (It should be noted that poor permeabilization and enzyme penetration resulted in only peripheral labeling around the perimeter of the nucleus, again underscoring the importance of internal controls for this technique). TUNEL labeling revealed that indeed only those taxol-treated cells blocked in prophase stained positively (Fig. 5 D–F). To ensure that the preferential staining of blocked prophase cells was not simply due to more efficient enzyme penetration in mitotic cells due to dissolution of the nuclear envelope and because the chromatin is condensed, we rigorously analyzed many fields of control cells and found all untreated mitotic cells at all stages of mitosis to be TUNEL negative (Fig. 5 A–C, indicated by m). Only the occasional dead cell stained positively in untreated populations (Fig. 5 A–C, indicated by arrowhead). It was also readily apparent that all M-blocked cells appeared to be positive even though 8 hr of treatment was well before the time cell death was apparent by Trypan blue staining or DNA ladders were evident by gel electrophoresis. TUNEL labeling and staining of the multimininucleated cells confirmed morphological observations of Hoechst-stained cells which indicated that these cells also progressed to an apoptotic pathway of cell death. However, multimininucleated cells only began to exhibit positive TUNEL staining from 3 days onwards after G1 arrest in a much more staggered fashion (Fig. 5 K and L; Hs578T cells are shown since multimininucleation was more common in these cells than in HeLa cells which have a rapid doubling time).
p53 Plays a Role in G1 Arrest Following Aberrant Mitosis but Does Not Affect Taxol-induced Cell Death Following Arrest in M
Multimininucleated wt p53 MEFs were never seen in mitosis throughout the detailed observations made during the subsequent 1- to 6-day period. Furthermore, those multimininucleated cells remaining attached at 6 days (Fig. 8D) were a similar size with similar chromatin content to multimininucleated cells fixed after 18 hr of taxol treatment (Fig. 8C) indicating arrest in G1. In contrast, multimininucleated p53−/− MEFs continued to enter mitosis but as a result of continued aberrant mitosis and lack of cytokinesis, they become progressively larger in size with a progressively greater complement of mini-nuclei (i.e., greater polyploidy; Fig. 8 E–H). Compare the size of the mitotic cell after 6 days taxol treatment in Panels E and F with the size of cells in mitosis in Panels A, B (control), and C (wt p53 after 18 hr). The cell indicated by the arrow in Panels G and H is just at the onset of chromosome condensation and so clearly reveals the condensation of all the individual mininuclei within the same cell, confirming the entry of multimininucleated cells into M 6 days after taxol treatment. In addition compare the considerably larger size of the multimininucleated cell to the lower left of the cell entering M indicated in Panels G and H, seen after 6 days, to the size of the multimininucleated wt p53 cells seen after both 18 hr and 6 days shown in Panels C and D, respectively. FACS analysis was carried out with propidium iodide stained cells that had been fixed after 0 hr, 24 hr, and 72 hr of taxol treatment. This confirmed that only the p53-null MEF population proceeded past the 4N DNA compartment after 24 hr and 3 days of taxol treatment. By 3 days, 70% of the p53-null MEFs were seen to be in the 8N and greater DNA compartment compared with 10% of untreated cells. In contrast, wt p53-expressing MEFs showed no such trends (data not shown; similar to trends shown by Cross et al. in Ref. 75 for MT destabilizing drugs).
Hoechst-staining revealed that multimininucleated MEFs expressing wt p53 developed all the morphological characteristics of apoptosis between 4 and 5 days following G1 arrest: initially, the DNA began to develop more intense punctate staining under the nuclear membrane and subsequently the DNA became highly globular. Little evidence of such changes was observed in p53-null MEFs. TUNEL staining of the two lines confirmed this difference dramatically. As illustrated in Fig. 9, the multimininuclei in wt p53-expressing MEFs all stain very brightly by TUNEL labeling 3–6 days following taxol-induced G1 arrest (Fig. 9 A–C represents cells after 6 days taxol treatment). In contrast, the multimininuclei in p53-null MEFs remain negative after 6 days (Fig. 9 D–F).
Comparison of EC 50 values for taxol-induced mitotic effects and cytotoxicity between ±p53 MEF phenotypes
Mitotic Arresta (nM)
Aberrant Mitosisa (nM)
wt p53 MEF
135.6 ± 9.6
46.2 ± 5.3
95.4 ± 32
p53 −/− MEF
120.0 ± 6.1
47.2 ± 0.1
110.7 ± 17.4
As mentioned above, those MEFs blocked directly in prophase detached and became nonadherent. Although detachment and Hoechst staining were consistent with these cells being destined for apoptosis, they were too transitory to obtain clearcut morphological evidence of apoptosis. Therefore, we sought to obtain convincing evidence that taxol-induced, early-prophase blocked cells in both MEF lines had initiated apoptosis using TUNEL staining. Cells were fixed after 8–10 hr of exposure to 1 µM taxol, while the cells were still adherent (peak detachment of M-blocked cells usually occurred between 10 and 15 hr) for ease of processing for TUNEL. As shown in Fig. 11, prophase-blocked cells are seen to be TUNEL positive in both wt p53 (Fig. 11, C and D) and p53-null (Fig. 11 E and F) lines and compare with the negative staining of the untreated mitotic cells (Fig. 11, A and B). Also note the multimininucleated cell at the bottom of Panels C and D which is TUNEL negative at this time point.
The data presented above clearly indicates that taxol-induced cytotoxicity correlates with perturbation of mitosis and cell cycle arrest for both the carcinoma lines and the normal HS578Bst line we studied. Our data supports the proposals first made by Kung et al. (Ref. 20, see also Refs. 19 and 23) and Jordan et al. (18) that taxol-induced cytotoxicity correlates with selective perturbation of mitosis rather than gross changes in interphase arrays as proposed by Rowinsky et al. (8) based on their studies of taxol effects on leukemic lines. We detected two forms of taxol-induced cell cycle arrest: arrest in prophase at the G2/M transition and, for those cells that succeeded in passing through this block and underwent aberrant mitosis; arrest in the subsequent G1 stage. For all the human cell lines that we examined, the major fraction of the population entering M during the time period taxol remained effective became arrested in prophase, with only a minor fraction becoming arrested in G1 as multimininucleated cells. As noted previously by others, we found rodent cells to be more tolerant of mitotic arrest per se than human lines (5,6,18,23). Rather the majority of the population proceeded through a dysfunctional mitosis to become arrested in G1 as multimininucleated cells (the restitution nuclei described by De-Brabander et al. in their studies on rodent PtK2 cells [5,6]). However, this species difference is a relative difference rather than an absolute one since we demonstrate here that rodent cells also displayed taxol-induced arrest in early M but at higher taxol concentrations than the EC50 for aberrant M and subsequent G1 arrest. As we have shown, this can be readily missed due to the transitory nature of the detached prophase-blocked cells due to phagocytosis by the adherent interphase cells. Both forms of cell cycle arrest led to cell death which by all morphological and DNA gel criteria occurred by apoptosis irrespective of the species cell type studied.
Our morphological and kinetic studies indicated that cell death occurred more rapidly in those cells arrested in M. Multimininucleated cells died with much slower kinetics over 3–7 days following taxol treatment. Those interphase cells that did not passage M during the time period taxol remained effective (within 48 hr), appeared to survive and subsequently proliferate normally. We employed the TUNEL method for in situ DNA end labeling to accurately determine the time course for induction of apoptosis following both types of cell cycle arrest, since classical DNA ladders were only detectable after 24 hr. TUNEL labeling revealed not only that the onset of DNA nicking occured first specifically in those cells arrested in prophase but also that this occurred surprisingly rapidly; within 30 min following taxol-induced arrest in prophase. In contrast, those cells that progressed through aberrant mitosis and were arrested in the subsequent G1 stage, became TUNEL positive only 2–4 days following G1 arrest. This was similar for all the human cell types examined, as well as Rat 1 (data not shown) and wt p53-expressing MEFs.
The majority of commonly used cancer therapeutic agents, including γ-irradiation, cause DNA damage and have been shown to exert their cytotoxic effects by triggering apoptosis (35–43). DNA nicking has been shown to directly elevate p53 protein expression which in turn induces arrest in the subsequent G1 or G2 stage of the cell cycle and then activates apoptosis by an as yet uncharacterized pathway (43,44). Elevated p53 levels directly up-regulate expression of the broad specificity cyclin-cdk kinase inhibitor, WAFl/Cipl/p21 (45–48,54,74) which appears to mediate the p53-dependent inhibition of cyclin-dependent kinase activities in G1 following γ-irradiation (45,47,54,62). The G2 block induced by many DNA-damaging antineoplastic agents entails a similar p53-mediated up-regulation of WAFl/Cipl/p21 (41,54).
Since taxol exhibits a completely different mechanism of action to the other antineoplastic agents, binding specifically to and thereby stabilizing intracellular MTs (1–4), which in turn induces predominantly arrest in M (1,3,26), we examined the role of p53 in taxol-induced apoptosis. For this we chose to compare MEFs expressing wt p53 with MEFs with a null p53 phenotype for an unambiguous answer. As expected, p53 status did not affect the primary consequences of taxol treatment. Taxol induced the same aberrant spindle formation in both wt p53 and p53-null MEFs. This led to a subset (∼1/3) of cells exhibiting true arrest in prophase at relatively high taxol concentrations (EC50 ∼ 130 nM) and the remainder proceeding through aberrant mitosis to become multimininucleated cells (EC50 ∼ 46 nM). However, whereas the wt p53-expressing MEFs became arrested in this first G1, the p53-null MEFs failed to arrest. They continued to progress through several rounds of the cell cycle in the absence of cytokinesis, becoming progressively larger with a progressively larger complement of mininuclei. Detailed morphological studies by Hoechst-chromatin staining of treated cell populations strongly suggested the wt p53-expressing multimininucleated cells became immediately arrested in G1 since the chromatin content remained identical from the time these aberrant cells first appeared (i.e., bona fide G1) to all subsequent time points. This was confirmed by FACS analysis. Studies on similar p53 +/+ and p53 −/− cell lines by Cross et al. (75), but using the MT-destabilizing agents nocodazole and colcemid, gave FACS DNA content data that was identical to the data we obtained with taxol-treated MEFs. Both by morphological criteria and by TUNEL labeling it was evident that multimininucleated wt p53-expressing MEFs developed all the hallmarks of apoptotic cell death. In contrast, the multimininucleated p53-null MEFs failed to exhibit signs of apoptotic death throughout the following 7-day period. Despite this, higher taxol concentrations induced a similar decline in total viable cell number in both lines rather than exerting solely a cytostatic effect in the p53 null line as might be predicted from the above observations. Detailed morphological characterization throughout the time course of the first cell cycle (∼15–18 hr) revealed that in fact this decline in viable cells correlated with the EC50 for arrest in prophase which was similar for both MEF lines. TUNEL staining of the two MEF lines confirmed that a subset of cells seen in prophase after 10 hr of taxol treatment did initiate DNA nicking, indicative of the induction of apoptosis.
Taken together, our results indicate that the antineoplastic mechanism of action of taxol evokes two distinct apoptotic pathways. The first is triggered by arrest in prophase at the G2M transition which leads to the rapid activation (within 30 min) of a p53-independent apoptotic pathway. The second, manifested by those cells that succeed in progressing through an aberrant mitosis, is a p53-dependent arrest in G1 which leads to a slower p53-dependent apoptotic pathway. We observed G1 arrest at taxol concentrations around the EC50 values for mitotic effects in all the human lines, albeit for a very small percent for the faster growing HeLa and Hs578T cells. Yet, Hs578T cells have been shown to harbor mutant p53 carrying a point mutation (67). Since mutant forms of p53 have been shown to have potential “gain of function” activity (63–64), it is clear that p53 protein carrying a point mutation is not synonymous with a null phenotype. Recently, it has been shown that antineoplastic agents may still be effective at inducing up-regulated expression of WAF1/Cip1/p21, the downstream effector of p53-mediated G1 or G2 arrest, in cell lines carrying point mutations in p53 (77).
The study by Cross et al. (75), which appeared during the final preparation of this manuscript, investigated the effects of the MT-destabilizing drugs, nocodazole and colcemid, which also perturb mitosis, on p53+/+ and p53-null MEFs. Although the data convincingly showed that wt p53 MEFs became arrested with 4N DNA content, but p53-null MEFs failed to arrest and accumulated progressively increasing amounts of DNA, the authors concluded that this data indicated that p53 was essential for a spindle checkpoint in M. However, the 4N DNA compartment was not subdivided into normal G2, cells in M and multimininucleated cells. Therefore, the data could indicate equally well that only the arrest in G1 following aberrant mitosis is compromised by the absence of p53. The detailed microscopic analyses we present here, clearly show that for taxol concentrations ≥100 nM, nearly all cells remaining after 24 hr are multimininucleated cells, so represent cells that have passaged aberrant mitosis to become aberrant 4N DNA-containing cells in G1. Since MT-destabilizing drugs effect a similar M and G1 block to taxol (18–20,22), it is clear the accumulation of cells in the 4N DNA compartment observed by Cross et al. (75) actually represent multimininucleated cells after 24 hr. By accounting for the fate of all the cells, which revealed the loss of M-blocked cells by phagocytosis, and titrating a taxol concentration that can effect a block in M at prophase in these murine cells, the data presented here in this report indicates that in fact the mitotic checkpoint is independent of p53 status. Our preliminary observations indicate that cyclin B levels are higher in taxol-blocked prophase cells than in normal cells in prophase through metaphase (data not shown). Further experimentation is required to determine whether this is indicative of heightened p34cdc2/cyclin B kinase activity. However, these observations are interesting in light of the following observations: (1) prolonged p34cdc2/cyclin B kinase activity might underly colcemid cytotoxicity (45,46); (2) an increase in p34cdc2/cyclin B kinase activity is observed just prior to cell death after cisplatin-induced G2 block (i.e., downstream from the initial p53-WAF1/Cip1/p21 response effecting the G2 arrest [39,41]); (3) initiation of apoptosis involves p34cdc2 kinase activation (78) and up-regulation of cyclin A (79,80); and (4) deletion of the wee1 kinase responsible for maintaining the p34cdc2 kinase in a phosphorylated inactive form (81) induces a “mitotic catastrophe” state that bears all the morphological features of taxol-blocked apoptotic cells in prophase (82). It remains to be shown whether, indeed, arrest in M directly activates a p34cdc2 kinase apoptotic response, in comparison with the p53-WAFl/Cipl/p21-dependent response that effects G1 block by inhibition of cyclin/cdk kinase activities well in advance of the active process of apoptosis.
Both p53-dependent and -independent pathways of apoptosis have been shown exist. While the former clearly operate in response to DNA damage resulting from γ-irradiation and DNA damaging agents (35–43), apoptotic pathways operating in normal development resulting from growth factor deprivation function through p53-independent pathways (52,54,68). Our data suggest that direct block in prophase also bypasses a p53-WAF1/Cip1/p21-dependent pathway. This could provide some explanation as to why taxol, uniquely among all the chemotherapeutic agents utilized to date, has shown efficacy against advanced stage, drug refractory leukemia, ovarian, and metastatic breast cancers (9–12). A high proportion of many types of advanced solid tumor types express mutant p53 (55–59,63,64). Thus, the commonly used cancer therapeutic agents that induce DNA damage and thereby directly activate the p53-dependent apoptotic pathway would be predicted to be ineffective against late-stage tumors consisting of clonally expanded, p53 mutant-expressing cells. By inducing a rapidly activating, M-block-dependent apoptotic pathway that is p53-independent (and presumably WAF1/Cip1/p21-independent), taxol may therefore bypass at least this one common pathway of aquired resistance to antineoplastic agents. The MT destabilizing drugs (e.g., vinblastine, vincristine, colchicine, podophyllotoxin) also appear to induce cell cycle arrest specifically in M, but to date their use in the clinic has been restricted by a poor therapeutic index. Although taxol appears to have an improved therapeutic index and to be efficacious for a broader spectrum of cancer types, its use is still limited by its poor solubility and side effect profile. Therefore, the discovery of additional structural classes of antineoplastic agents that exhibit a mechanism of action similar to that of taxol and specifically induce arrest in M-phase (73), but have the potential for an improved side effect and solubility profile, might prove highly useful in the fight against cancer.
We greatly appreciate the assistance of Judy Miller in carrying out FACS analysis and also thank J. Xu for helpful discussions on the TUNEL method and Dr. Helen Ranney for valuable input during the completion of this manuscript.
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