Introduction

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Antimitotic drugs are widely used in chemotherapy. They usually target the tubulin/microtubule network of the cytoskeleton, which is formed by an assembly of cytoplasmic tubulin dimers. Several synthetic and natural compounds interact specifically with tubulin and microtubules, fundamentally destroying their dynamic character and leading to cell death. A large number of these agents are plant-derived (Lin et al., 1988). Two Vinca alkaloids from Catharanthus roseus, vinblastine, and vincristine are widely used in cancer therapy (Rowinsky and Donehower, 1997). Both drugs inhibit the self-assembly of tubulin into microtubules at substoichiometric concentrations by forming a tubulin-drug complex at the end of a growing microtubule and thus blocking self-assembly (Wilson et al., 1976; Margolis et al., 1980). In addition, it is known that vinblastine induces G₂/M arrest and subsequent apoptosis in different cell lines (Fan et al., 2001). Although the primary target of these Vinca alkaloids is the microtubular network, we recently reported that they also bind to calmodulin and suspend its modulating effect (Molnár et al., 1995; Vértessy et al., 1998). Calmodulin is a ubiquitous Ca²⁺ receptor that is involved in cell proliferation and in the regulation of the cell cycle (Rasmussen and Means, 1989; Lu and Means, 1993), but also in other essential cell processes (Cohen and Kee, 1988). The addition of specific anticalmodulin drugs, such as W13, to cell cultures inhibits re-entry of growth-arrested cells into the cell cycle (G₀/G₁ transition), progression into and through the S phase, and entry and exit from mitosis (Sasaki and Hidaka, 1982; Chafouleas et al., 1984; Agell et al., 1998).

Numerous semisynthetic derivatives of Vinca alkaloids have been synthesized, in response to the extensive need for potent antimitotic agents in clinical chemotherapy, because vinblastine and vincristine have undesired side effects. We recently reported (Orosz et al., 1997a; Vértessy et al., 1998) that the new semisynthetic bis-indole derivative KAR-2, has high anti-microtubular and anti-tumoral activities and lower toxicity than the Vinca alkaloids used in chemotherapy. Moreover, it interacts with calmodulin in vitro but, in contrast to vinblastine and vincristine, it does not exhibit anticalmodulin activity in vitro enzymatic assays (e.g., phosphofructokinase) (Orosz et al., 1997a,b; Orosz et al., 1999). In addition, we reported the synthesis of other bis-indole derivatives, KAR-3 and KAR-4, which are analogs of vinblastine and vincristine, respectively (see Fig. 1). These compounds are also powerful antimicrotubular agents with lower anticalmodulin activity and toxicity than vinblastine or vincristine, although they are more toxic and no more powerful than KAR-2. (Orosz et al., 1999).

View larger version (18K): [in this window] [in a new window]   Fig. 1.   Structural formula of vincristine, vinblastine, and their bis-indole derivatives KARs.

In vertebrates, cell cycle is regulated by a family of Cdks formed by a catalytic subunit and a regulatory subunit called cyclin. Those kinases act sequentially during the cell cycle. Cdk4,6/cyclin D and Cdk2/cyclin E are essential during G₁ and G₁/S transition, Cdk2/cyclin A is active during S phase whereas Cdc2/cyclin A and Cdc2/cyclin B act during mitosis. Cylin A accumulates during S and G₂ phases and cyclin B during G₂ phase. During G₂ both cyclins bind to Cdc2. At G₂/M transition the complexes formed are suddenly activated by the Cdc25 phosphatase, which eliminates two inhibitory phosphates from the catalytic subunit. Cdc2 is responsible for nuclear lamina phosphorylation; consequently, nuclear envelope is disorganized during prophase. Cdc2 also phosphorylates histone H1 leading to chromatin condensation, which occurs at mitosis (Norbury and Nurse, 1992; Reed, 1992; Sherr, 1994; Morgan, 1997). Degradation of cyclin A and cyclin B during metaphase and anaphase, respectively, leads to Cdc2 inactivation and consequently to mitosis exit. When mitotic spindle is incorrectly formed, a checkpoint is activated that inhibits Cdc2 inactivation; consequently, mitosis exit and cell cycle progression. This checkpoint usually works by inhibiting degradation of mitotic cyclins A and B (Gong et al., 1995; Rudner and Murray, 1996). The objective of the present article is to characterize the effect of KARs at cellular levels compared with that of vinblastine and vincristine to asses the origin of the distinct behaviors manifesting in in vitro and in vivo conditions.

The fact that KARs are powerful antimicrotubular agents with lower anticalmodulin activity and toxicity than vinblastine or vincristine makes them promising antitumoral agents, especially KAR-2, which is the less toxic (Orosz et al., 1999). In this article, we compare the effects of KAR2, with those of some other bis-indole derivatives, on human neuroblastoma cell line (SH-SY5Y) at different levels. Analysis of DNA content by flow cytometry showed that treatment with vinblastine and vincristine caused a partial arrest in both G₀/G₁ and G₂/M phases, whereas KAR-2 specifically arrested cell cycle at G₂/M phase. Analysis of chromosome condensation and phospho-H3 (Hendzel et al., 1997), cyclin A and cyclin B1 expression levels, Cdc2 activity and microtubular network, showed that KAR-2-treated cells were arrested in mitosis with a majority of abnormal mitotic spindles. Interestingly, KAR-2 had no effect on interphase microtubules, whereas vinblastine partially destroyed the microtubular network of interphase cells on neuroblastoma cell line.

	Materials and Methods

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Cell Culture. Human neuroblastoma SH-SY5Y cells (a generous gift from Dr. Jacint Boix, University of Lleida, Spain) were cultured in Dulbecco's modified Eagle's medium (Biological Industries, Kibbutz Beit Haemek, Israel) supplemented with 10% fetal calf serum (Biological Industries), and antibiotics: 100 U/ml penicillin and 100 µg/ml streptomycin (Invitrogen, Carlsbad, CA). Cells were grown at 37°C under an atmosphere of 5% CO₂. SH-SY-5Y cells were seeded in six-well plates, 4 × 10⁵ cells/well, for all the experiments except where indicated. The final volume in all the dishes was 2 ml. Compounds to be tested were added to cultures 1 day after seeding to ensure uniform attachment of cells at the beginning of the experiments. The cell line used in this study was free of mycoplasma infection as shown by Gen-Probe Mycoplasma Tissue Culture NI Rapid Detection System (Fisher Scientific, Pittsburgh, PA)

Bis-Indole Derivative Treatment. The final concentrations of compounds used were: KAR-2 at 316 nM, KAR-3 at 316 nM, KAR-4 at 355 nM, vincristine at 14 nM, and vinblastine at 5 nM. These concentrations of each compound have been determined by microculture tetrazolium assay as equivalent in causing 50% of inhibition of cell growth in a previous study in 72 h (Orosz et al., 1999). Stocks were dissolved in phosphate-buffered saline (PBS) at 1 mg/ml for each compound and stored at 20°C. On the day of the experiment, compounds were diluted in culture medium. .

Inhibition of Cell Growth and Cell Cycle Analysis. Plasma membrane integrity was estimated by FACS analysis using two different methods. In the PI (Sigma Co, St. Louis, MO) staining method without cell permeabilization, cells were incubated with 18 µg/ml PI and 5 µg/ml RNase (Roche Molecular Biochemicals, Mannheim, Germany). The fluorescence of cells was analyzed by flow cytometry using an Epics XL flow cytometers (Beckman Coulter, Fullerton, CA). The second methods was FITC-Annexin V/PI double staining (Genzime, Cambridge, MA) (see Assessment of Apoptosis).

Assessment of Apoptosis. Cells were seeded in six-well plates, 25 × 10³ cells/well and, after a pre-equilibration period of 24 h, they were exposed to the bis-indole derivatives to be tested for a period of 24 or 48 h. Double-staining for FITC-Annexin V binding and for cellular DNA using PI was performed according the product insert. Cells were processed by flow cytometry. Approximately 3 × 10⁴ cells were measured for each histogram.

Gel Electrophoresis, Immunoblotting, Immunoprecipitation, and Protein Kinase Assay. Cyclin A and cyclin B1 were analyzed using electrophoresis and immunoblotting, as described previously (Taulés et al., 1998). Monoclonal antibodies against cyclin A (Santa Cruz Biotechnology, Santa Cruz, CA) were used at 1:100 dilution, antibodies against cyclin B1 (Upstate Biotechnology, Lake Placid, NY) at 1:500 dilution. As a control of protein loading the blot membrane was also hybridized with polyclonal anti-Cdk4 at 1:500 dilution. The reaction was visualized with 5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium (Promega, Madison, WI). The experiment was done at 24 h of incubation with the tested compounds. To determine Cdc2 activity levels, immunoprecipitations were performed as described previously (Taulés et al., 1998), except that 3 µg of anti-Cdc2 antibodies (Upstate Biotechnology) were used and 2 µg of histone H1 was used as substrate.

Immunocytochemistry. Cells were grown on glass coverslips coated with poly(D-lysine) (50 µg/ml at final concentration) (Sigma). For double-labeling, anti--tubulin (Roche) and PI were fixed in cold methanol for 2 min. After three washes in sterile PBS, cells were incubated 1 h at room temperature in a humidified atmosphere, with the specific monoclonal antibody anti--tubulin (1:50 dilution) with 1% ovalbumin. Coverslips were then washed three times (5 min each) in PBS and incubated for 1 h at room temperature in the dark with FITC-coupled anti-mouse-IgG secondary antibody (1:100 dilution; Promega). After three washes in PBS, RNase was added at a final concentration of 100 µl/ml for 30 min at room temperature in the dark and DNA stained with PI (1 µg/ml) for 1 min at room temperature in the dark. Cells were washed once in 0.1% PBS with Triton X-100 and twice in PBS. Finally, coverslips were mounted on glass slides with mowiol (Calbiochem, San Diego, CA).

	Results

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Inhibition of the Cell Growth by the New Vinca Alkaloids, KARs. Cell cultures with the same number of cells were treated separately with vincristine (14 nM), vinblastine (5 nM), KAR-2 (316 nM), KAR-3 (316 nM), and KAR-4 (355 nM). These concentrations caused 50% inhibition of cell growth as determined by microculture tetrazolium assay (Orosz et al., 1999). The number of cells was counted at different times (8, 24, 48, and 72 h) after treatment and plotted against the percentage of initial number of cells (0 h). It was observed that all the compounds induced a similar time-dependent decrease in cell growth (Fig. 2). The effects of all these compounds became significant 24 h after the beginning of the treatment. Between 24 and 48 h, the decrease in cell growth was approximately 50%, and less than 13% of the cells survived after 72 h (Fig. 2). These results corroborate that the different concentrations of each compound selected for this study cause an equivalent mortality of cells at different times. Thus, these concentrations (which we will refer to as IC₅₀ values) are suitable for the comparative studies reported in the following sections.

View larger version (26K): [in this window] [in a new window]   Fig. 2.   Time-dependent antiproliferative effects of vinblastine (VBL), vincristine (VCR), and the new KAR bis-indole derivatives on neuroblastoma SH-SY5Y cells. Cells (4 × 105 cells/well) were incubated for 0 to 72 h with the bis-indole derivatives. Every 8, 24, 48, and 72 h, the wells were trypsinized and cells were counted in a XL Coulter FACS. Cell numbers represent only viable cells, as measured by the exclusion of PI and the percentage is relative to the initial cell number. Results are given as mean ± S.E.M. of three independent experiments performed in triplicate. Control, ; KAR-2, 316 nM (); KAR-3, 316 nM (); KAR-4, 355 nM (); VBL, 5 nM (); VRC, 14 nM (). A, control and KAR-2. B, control and KAR-3. C, control and KAR-4. D, control, vincristine and vinblastine.

Treatment with KAR-2, KAR-3 and KAR-4 Completely Arrested Neuroblastoma Cells in G₂/M Phase of the Cell Cycle, with a Low Percentage of Apoptosis. The experiment was performed with asynchronously growing cells. In this population (0 h), most cells were in G₁ phase (48.7% ± 0.8%) or S phase (41.4% ± 1.1%), whereas 9.9% ± 0.5% were in the G₂/M phase (n = 37; mean ± S.E.M). To examine the effects of the cytotoxic agents at their IC₅₀ values on cell cycle distribution, neuroblastoma cells, treated with each compound for 8, 24, and 48 h, were analyzed by FACS (Fig. 3). Treatment with vincristine or vinblastine led to a different pattern of cell cycle distribution than treatment with KAR-2, KAR-3, or KAR-4. After 8 h of treatment, KAR-2, KAR-3, and KAR-4, similarly to vincristine and vinblastine, induced a small but significant decrease in the percentage of cells in G₁ phase and a small increase in percentage of cells in G₂/M and S phases with respect to nontreated cells. At 24 h of treatment with KARs, 65% of treated cells were in G₂/M phase, whereas less than 8% of cells remained in G₁. The percentage of cells in S phase increased with respect to control cells, except in the case of KAR-2-treated cells, which were not significantly different from control cells. In contrast, when cells were treated 24 h with vincristine or vinblastine, less than 50% of cells were in G₂/M and 36% of cells remained in G₁. The percentage of cells in S phase decreased for vinblastine and remained statistically unchanged with respect to control for vincristine.

View larger version (38K): [in this window] [in a new window]   Fig. 3.   Cell-cycle phase distribution in neuroblastoma cell line after vinblastine, vincristine, KAR-2, KAR-3, and KAR-4 treatment. Gray bars represent treated control tumor cell cycle frequency distribution (y-axis) expressed as percentage of sorting into the G0-G1, S, and G2-M phases. The other bars represent KAR-2 (316 nM), KAR-4 (355 nM), vinblastine (5 nM), KAR-3 (316 nM), and vincristine (14 nM) treatment. After 8 h of treatment, cell populations were decreased in G0/G1 phases, whereas there was a small increase in both the S and G2/M phases [n = 12, values represent mean ± SD., *, significantly different (P < 0.01)]. After 24 h of treatment, cells treated with KARs, vincristine, and vinblastine exhibited a decrease in G0/G1 phase, whereas there was an increase in the G2/M phase; however, the effect of KARs was significantly different from that of vinblastine and vincristine (n = 12; mean ± SD.; *, P < 0.01). After 48 hr of treatment, cells treated with KARs, vincristine, and vinblastine exhibited a decrease in G0/G1 phase, whereas there was an increase in the G2/M phase. Although KARs caused an irreversible decrease in G2/M, the decreases caused by vincristine and vinblastine were reversible (n = 12; mean ± S.D.; *, P < 0.01).

View larger version (17K): [in this window] [in a new window]   Fig. 4.   Cell cycle analysis of neuroblastoma cells untreated or treated with KAR-2 (316 nM), W13 (30 µM), KAR-2 and W13 (316 nM and 30 µM, respectively), and vinblastine (VBL; 5 nM) for 24 h. DNA was stained with PI as described under Materials and Methods

View this table: [in this window] [in a new window]   TABLE 1 Cytometric analysis of Annexin V-FITC staining and PI accumulation after exposure to the bis-indole derivates Final concentrations, KAR-2, 316 nM; KAR-4, 355 nM; VBL, 5 nM; VRC, 14 nM; KAR-3, 316 nM for 48 h on neuroblastoma cells. Apoptotic cells (annexin V+/PT), necrotic cells (annexin V+/PI+) include late apoptotic cells. 30,000 cells were counted. Data are shown as the mean value ± S.E.M. (Annexin V-AV).

Treatment with KARs Induced an Increase of the Number of Cells with Condensed Chromosomes and with Phospho-H3. To determine whether cells were stopped in G₂ or in mitosis after treatments, chromatin was visualized by Hoechst staining. Results showed that only 3.4% ± 0.6% of control cells had condensed chromosomes. After 24 h of treatment with the compounds, cells with condensed chromosomes were the following: 46 ± 6% after KAR-3, 41 ± 2% after KAR-2, 36 ± 5% after KAR-4, 34 ± 3% after vincristine, and 18 ± 2% after vinblastine. To confirm that the cells were really stopped in mitosis after the different treatment, we analyzed phosphorylation of H3 with a specific antibody. H3 is phosphorylated at the beginning of metaphase and until telophase. Thus, it can be used as a mitotic marker. After 24 h, phosphorylated H3-positive cells were the following: 10.7 ± 3% without treatment, 46.4 ± 7% after KAR-2, 16.3 ± 1%. These results correlated with the condensed chromosome quantification shown above. These percentages correlate with the results of cell cycle analysis. However, it should be taken into account that the percentage of cells with phospho-H3 or condensed chromosomes is always lower than the percentage of cells arrested in G₂/M (by FACS), because the cells in G₂ do not have condensed chromosomes or phosphorylated H3. Thus, KARs induced a strong arrest of cells in mitosis, which is consistent with their inhibitory effect on tubulin polymerization and mitotic spindle formation.

Effect of KARs on the Levels of Cyclins A and B1 and Cdc2. In cells with normal spindle checkpoint, failure of correct mitotic spindle formation should prevent mitotic exit by inhibition of cyclin A and B1 degradation, and thus inhibition of Cdc2 inactivation. Consequently, we analyzed the levels of cyclins A and B1 and activity of Cdc2 after the various treatments. Western blot analysis showed that all bis-indole derivative-treated cells had higher levels of cyclin A than nontreated cells. Furthermore, the levels of cyclin A were slightly higher after KARs treatment compared with vinblastine or vincristine (Fig. 5.1). Cyclin B1 levels were similar in vinblastine- and vincristine-treated cells than in control, whereas the levels in KAR-2-, KAR-3-, and KAR-4-treated cells were higher. Furthermore, we found that Cdc2 activity was higher in Kar-2 or vinblastine-treated cells than in nontreated cells. The activity after Kar-2 treatment was double that after vinblastine treatment (Fig. 5, 2). These results indicate that the arrest induced by KAR-2 occurred before anaphase-telophase transition and that this arrest was stronger in KAR-2- than in vinblastine-treated cells.

View larger version (51K): [in this window] [in a new window]   Fig. 5.   Effect of vinblastine (VBL), vincristine (VCR), KAR-2 (K2), KAR-3 (K3), and KAR-4 (K4) on the levels of cyclins and Cdc2. 1, determination of cyclin A (A) and cyclin B1 (B1) levels by immunoblotting after treatment with the anti-cancer drugs for 24 h as indicated under Materials and Methods. 2, determination of the level of Cdc2 after treatment with VBL or KAR-2. Treated and nontreated cells were lysed at 24 h. Cell lysates were immunoprecipitated with antibody against Cdc2. Immunoprecipitations using normal rabbit serum were performed as controls. To analyze the activity of Cdc2, histone H1 substrate was used.

KAR-2 Induced Aberrant Spindle Formation but Did Not Affect Interphase Microtubules. To further characterize the mechanisms associated with KAR-2-induced growth arrest, we examined the effects of KAR-2 at concentrations of 316 nM and 5 nM on spindle and microtubules behavior. The effects were compared with those induced by vinblastine at 5 nM and 316 nM. Immunostaining with anti--tubulin antibody revealed marked differences between the control and vinblastine or KAR-2 treated cells (Fig. 6). Mitotic spindles were normal in untreated cells. As shown above, in cells treated with vinblastine at 5 nM, the number of cells with condensed chromosomes and phospho-H3 were higher than in untreated cells. Although some of them had mitotic spindle with normal appearance (approximately 30% of mitosis was normal), the microtubule network of most of the interphasic cells was severely damaged. In contrast, KAR-2-treated cells (at 316 nM) had a high number of aberrant mitotic spindles (less than 10% of mitosis were normal), whereas the interphase cells had an apparently normal microtubular network (Fig. 6). KAR-2, at a concentration of 5 nM, had no apparent effect on either the interphase or mitotic microtubules. In contrast, vinblastine, at a concentration of 316 nM induced complete destruction of the microtubular network of interphase cells (data not shown).

View larger version (31K): [in this window] [in a new window]   Fig. 6.   Confocal Microscope images of control (C), KAR-2- (K2), and vinblastine- (VBL) treated cells at 316 and 5 nM, respectively. Cells were subcultured on coverslips. After 24 h of treatment, cells were fixed as described under Materials and Methods. Tubulin was visualized by immunofluorescence after treatment with anti--tubulin (primary) and FITC-labeled (secondary) antibodies (green). DNA was visualized by PI staining (red). I, interphasic cells; M, mitotic cells; example of normal anaphase (left) and metaphase (right). K2, example of the predominant mitotic spindle; VBL, example of normal mitotic spindle.

	Discussion

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Bis-indole derivatives extracted from Catharanthus roseus such as vincristine and vinblastine have been used extensively as antimitotic drugs in cancer chemotherapy since the 1960s. We reported the synthesis and characterization of novel bis-indole compounds, KAR-2, KAR-3, and KAR-4, which are semisynthetic derivatives of bis-indoles occurring in Catharanthus roseus extract in relatively large amounts (Orosz et al., 1997a,b, 1999). The most promising candidate to be used as an antitumoral drug was KAR-2, which has similar or even higher anti--tubulin and antitumor activity compared with other bis-indoles but significantly lower toxicity (Orosz et al., 1997b, 1999). We have recently documented that although their antimicrotubular activities in in vitro systems are similar, KAR-2 and vinblastine display distinct effects in neuroblastoma and other cell lines (primary brain cells, PC12, Ehrlich ascites tumor cells) (Orosz et al., 1999) and in mice hosting different tumor cells. Concerning the in vivo effect of KAR derivatives versus vinblastine and vincristine in tumor cells, we have also demonstrated that maximal cytotoxic activities of KARs in mice hosting leukemia P388 or Ehrlich ascites tumor cells are similar to those of vinblastine and vincristine; however, significant prolongation of life span could be reached with KAR derivatives after the administration of a single dose (Orosz et al., 1999). The single dose administration is an important issue in relation to the multidrug resistance problem known in the case of vinblastine and vincristine. In addition, the KAR-2 administration did not induce neurotoxic side effects (e.g., paralysis of bladder or lower extremities) as observed with bis-indoles routinely used in therapy. In this study, we further characterized the effects of KAR-2 and its parent compound in cultured neuroblastoma cell line as a model to better understand the cellular mechanism of KARs compared with vinblastine and vincristine.

FACS analysis showed that KARs completely inhibit cell cycle progression in G₂/M phases, in contrast to vincristine and vinblastine, which caused partial arrest in G₀/G₁ and G₂/M phases. The results obtained on cell cycle arrest with vincristine and vinblastine are consistent with those obtained with these drugs in other cell lines (Jordan et al., 1992; Wilson and Jordan, 1994; Molnár et al., 1995; Petru et al., 1995). It has been reported that calmodulin participates in the regulation of the G₀/G₁ transition (Chafouleas et al., 1984), the progression into and through S phase (Sasaki and Hidaka, 1982; Chafouleas et al., 1982; López-Girona et al., 1992), and the initiation and the exit of mitosis (Chafouleas et al., 1982). Accordingly, we suggest that these differences with KARs could be explained, taking into account that vinblastine and vincristine present anticalmodulin activity and that KAR-2 binds to a different site on calmodulin than vinblastine (Vértessy et al., 1998) and thus does not display anticalmodulin activity (Orosz et al., 1997a). Moreover, cell cycle perturbations observed when we treated neuroblastoma with a mixture of KAR-2 and W13 (a calmodulin inhibitor) were similar to those obtained with vinblastine alone. Although these results agree with our hypothesis that the effect of vinblastine on the cell cycle is a result of the combination of its antimicrotubular activity and its anticalmodulin activity, other possibilities could be considered to explain differences between vinblastine and KAR-2.

Because exposure of cells to KARs leads to higher levels of cyclin A and B1 than exposure of cells to vinblastine and vincristine, we concluded that KARs caused stronger mitotic arrest and seem to be more specific antimitotic agents than vinblastine or vincristine. This is corroborated by the fact that Cdc2 activity doubled in KAR-2-treated cells with respect to vinblastine-treated cells. KARs do not display polyploidy at 72 h, which is a desirable characteristic for a drug to be used in antitumor chemotherapy and also indicates that neuroblastoma cells activate the mitotic spindle checkpoint in response to KARs treatment. The cell cycle phase arrest specificity of antitumor drugs is important in oncology in developing clinical treatment protocols and designing antitumor strategies involving specific drug combinations. For instance, Stone et al. (1996) demonstrated that drugs that induce overexpression of p16 make normal cells more resistant than cancer cells to antimitotic drugs, because normal cells respond to p16 overexpression reversibly by arresting at G₁, whereas in many tumors, the p16 regulatory pathway is inactivated and thus cells progress to mitosis, where they become susceptible to antimitotic drugs. Testing several anticancer agents, they concluded that the most dramatic effect was observed with vinblastine and suggested that other agents more specifically directed against the G₂ or M phases than vinblastine might be more effective (Stone et al., 1996). From this point of view, the new KAR family of Vinca-alkaloids could be an excellent candidate for this therapeutic approach; this will be one of the objectives of our future studies with these drugs. The ability of KAR-2 to induce the formation of aberrant mitotic cells without any apparent destruction of the microtubular network of interphasic cells at their IC₅₀ level is in marked contrast to its mother compound vinblastine. These differences can be very important in a possible selective effect of KAR-2 against dividing cells, which is a desirable characteristic in any antitumoral drug. These differences can also be related to the lack of anticalmodulin activity of KAR-2; it has been reported that some of the microtubule-associated proteins are calmodulin-binding proteins (Ortega-Perez et al., 1994; Gonzalez et al., 1995).

We found that KARs, like vinblastine and vincristine, cause apoptosis rather than necrosis in human neuroblastoma cell line SH-SY5Y. However, the percentage of apoptotic cells was always low, which is consistent with the percentages of apoptosis after vincristine treatments reported in the literature for other cell lines (Tsurusawa et al., 1997). This finding is consistent with the higher percentages of mitotic cells after treatment of these compounds and with the evidence that cells may undergo apoptosis at any phase except mitosis (Pittman et al., 1994). In addition, it has been suggested that tubulin reorganization may play a role in the apoptotic process (Cotter et al., 1992).

Thus, the combination of the three differential characteristics of KAR-2 with regard its parental compounds vinblastine and vincristine, the fact that it completely arrests cell cycle at mitosis, and its capacity to generate aberrant mitosis without affecting interphase microtubules makes this compound particularly interesting from a pharmaceutical point of view.