Paclitaxel induces calcium oscillations via an inositol 1,4,5-trisphosphate receptor and neuronal calcium sensor 1-dependent mechanism

  1. Wolfgang Boehmerle*,†,
  2. Ute Splittgerber‡,
  3. Michael B. Lazarus‡,
  4. Kathleen M. McKenzie‡,
  5. David G. Johnston*,
  6. David J. Austin‡, and
  7. Barbara E. Ehrlich*,§
  1. Departments of *Pharmacology and
  2. ‡Chemistry, Yale University, New Haven, CT 06520; and
  3. †Neuroscience Research Centre, Charité Universitaetsmedizin Berlin, 10117, Berlin, Germany

  1. Edited by Solomon H. Snyder, Johns Hopkins University School of Medicine, Baltimore, MD, and approved October 4, 2006 (received for review August 23, 2006)

 
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Abstract

Taxol (Paclitaxel) is an important natural product for the treatment of solid tumors. Despite a well documented tubulin-stabilizing effect, many side effects of taxol therapy cannot be explained by cytoskeletal mechanisms. In the present study submicromolar concentrations of taxol, mimicking concentrations found in patients, induced cytosolic calcium (Ca2+) oscillations in a human neuronal cell line. These oscillations were independent of extracellular and mitochondrial Ca2+ but dependent on intact signaling via the phosphoinositide signaling pathway. We identified a taxol binding protein, neuronal Ca2+ sensor 1 (NCS-1), a Ca2+ binding protein that interacts with the inositol 1,4,5-trisphosphate receptor from a human brain cDNA phage display library. Taxol increased binding of NCS-1 to the inositol 1,4,5-trisphosphate receptor. Short hairpin RNA-mediated knockdown of NCS-1 in the same cell line abrogated the response to taxol but not to other agonists stimulating the phosphoinositide signaling pathway. These findings are important for studies involving taxol as a research tool in cell biology and may help to devise new strategies for the management of side effects induced by taxol therapy.

Taxol (Paclitaxel) is among the most commonly used chemotherapeutic drugs in clinical practice for treatment of ovarian and breast cancer, non-small-cell lung carcinoma, and AIDS-related Kaposi's sarcoma, as well as skin, head, and neck cancers (reviewed in ref. 1). First reports of the antitumor activity of an isolate from the bark of the pacific yew Taxus brevifolia date back to 1964, but taxol's main mechanism of action, the promotion of tubulin polymerization and stabilization of existing microtubules, was not discovered until 1979 (2). Despite its well developed mode of action in microtubule stabilization (2), there is evidence that taxol exhibits non-microtubule-associated biological functions (3). Only a few taxol binding proteins other than tubulin have been found. Several heat shock proteins identified in macrophage cell lysates (4) and the antiapoptotic protein Bcl-2 have been shown to interact with taxol. The interaction with BCl-2 may be important for the proapoptotic effects of taxol (5). Taxol frequently induces additional side effects such as acute hypersensitivity reactions, cardiac conduction disturbances, and neurosensory symptoms (6). The etiology of these potentially dose- and therapy-limiting side effects is still poorly understood and difficult to explain with the known taxol interaction partners.

One interesting suggestion for understanding these side effects comes from the observation that taxol exerts effects on cytosolic Ca2+ signaling. When concentrations of 8,500 ng/ml (10 μM) taxol were applied, opening of the mitochondrial permeability transition pore was observed (7, 8). One caveat of these studies is that high concentrations of taxol were used, whereas in most clinical applications even the maximum plasma concentration does not exceed 3,600 ng/ml (4.3 μmol/liter) (9), and steady-state plasma concentrations are even lower with reported values between 85 and 850 ng/ml (10).

In this study we aimed to determine whether much lower concentrations of taxol could alter cytosolic Ca2+ signaling and, if so, to characterize the involved pathways. We found that taxol in submicromolar concentrations induced oscillatory changes in cytosolic Ca2+ in an inositol 1,4,5-trisphosphate receptor (InsP3R)-dependent manner. Because there is no direct interaction between tubulin and the InsP3R, we used a C-7 biotinylated taxol probe and a display cloning procedure (11) to investigate the possibility of non-tubulin taxol binding proteins. We have cloned a binding partner from a T7 bacteriophage human brain cDNA library. The isolated protein has been identified as neuronal Ca2+ sensor 1 (NCS-1), which is a member of a family of Ca2+ binding proteins (12) and has recently been shown to modulate InsP3R-dependent Ca2+ signaling (13). Intriguingly, taxol increased binding of NCS-1 to the InsP3R and short hairpin RNA (shRNA)-mediated knockdown of NCS-1 abrogated taxol-induced Ca2+ oscillations. These findings suggest the need for caution when using taxol in cell biological studies where it is often added for microtubule stabilization and visualization. Furthermore, these findings introduce a pathway for the understanding of side effects specific to taxol therapy and may contribute to the future development of more effective derivatives.

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Results

Effects of Low Taxol Concentrations on Intracellular Ca2+ in Human Neuroblastoma Cells.

We monitored intracellular Ca2+ changes in the human neuroblastoma cell line SH-SY5Y with the fluorescent dye Fluo-4/AM. The investigations reported here used taxol concentrations mimicking steady-state concentrations observed in patients (10). Addition of taxol at a concentration of 800 ng/ml (937 nM) evoked an increase in intracellular Ca2+, typically within the first 40 seconds after bath application. This initial increase was followed by subsequent Ca2+ increases, thus creating an oscillatory pattern (Fig. 1 A).

Fig. 1.
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Fig. 1.

Taxol induces Ca2+ oscillations independent of extracellular and mitochondrial Ca2+. (A) Representative normalized Ca2+ changes induced by 800 ng/ml taxol (arrow). (B) Taxol-induced oscillations are concentration-dependent with a calculated EC50 of 83 ng/ml (arrow). (C) Power spectral analysis of the cell shown in A reveals that the dominant peak of taxol-induced Ca2+ oscillations occurs at ≈12 mHz. (D–F) Ca2+-free solution (D) (10 mM EGTA), preincubation of cells with cyclosporin A (E), and treatment with the mitochondrial uncoupler FCCP (F) did not block oscillations induced by 800 ng/ml taxol (arrow). A, 10 μM ATP; T, 3 μM thapsigargin.


Because taxol was dissolved in a 1:1 mixture of cremophore EL and absolute ethanol, a formulation that has been suggested to have biological effects of its own (reviewed in ref. 14), we also tested the effects of the vehicle at the same concentration (0.4 μl/ml) as that used in the experiments testing the effect of taxol on Ca2+ signals. In the taxol group ≈50% of all cells oscillated upon treatment, which was significantly different from the vehicle group. Likewise, more cells responded with transients to taxol treatment compared with vehicle-treated cells, with the majority of cells not responding to vehicle alone (Table 1). Spontaneous activity of untreated cells [173/5] was not significantly different from vehicle treatment in any of the three categories, confirming that vehicle at the concentrations used in the present study does not have an effect on intracellular Ca2+ signaling.

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Table 1.

Percentage of cells responding to various treatments as described in Results


Next, we tested the effect of a range of taxol concentrations. At the lowest concentrations of 0.8 ng/ml and 8 ng/ml, oscillations were not observed (0.6 ± 0.6% [167/4] oscillating at 0.8 ng/ml, and 0.5% ± 0.5% [147/4] at 8 ng/ml). After addition of 80 ng/ml, 30 ± 10% [318/6] of the entire population of cells oscillated, a trend that continued at 200 ng/ml (39 ± 13% [199/5]) (P < 0.05). Application of 1,600 ng/ml taxol induced oscillations in 53 ± 10% [172/6] of all cells, which was not significantly different from treatment with 800 ng/ml, indicating that the response saturates when ≈50% of all cells oscillate. By using a sigmoidal fit to the data, the calculated EC50 for evoking an oscillatory Ca2+ response was 83 ng/ml taxol (Fig. 1 B).

To measure the regularity of the taxol-induced Ca2+ oscillations we used power spectral analysis as described previously (15). We found that the Ca2+ oscillations could be described adequately with one major peak (example in Fig. 1 C), indicating a regular oscillatory response. The average oscillation frequency was 12.8 ± 0.9 mHz (n = 191 cells).

Neither Extracellular nor Mitochondrial Ca2+ Is Required for Taxol-Induced Oscillations.

To determine the contribution of extracellular Ca2+ to the taxol-induced oscillations, cells were observed in a Ca2+-free solution (0 Ca2+ plus 10 mM EGTA added to the extracellular solution). This treatment did not abolish the initial response, but there was a slight, yet not significant, reduction in the percentage of cells producing an oscillatory response to 800 ng/ml taxol (Table 1). However, power spectral analysis revealed a significantly (P < 0.01) reduced mean oscillation frequency of 7.2 ± 0.7 mHz compared with measurements when extracellular Ca2+ was present. We also observed in several experiments a decreasing amplitude of the response (Fig. 1 D), which, together with the reduced oscillatory frequency, suggests that, although extracellular Ca2+ is not necessary for the initial response, it is required to maintain the response, presumably to prevent depletion of intracellular Ca2+ stores.

Because previous studies have shown a direct effect of high taxol concentrations on mitochondrial permeability (7, 8), it was important to determine whether this observation would be obtained with the much lower concentrations used in this study. Cells were preincubated for >5 min in 5 μM cyclosporin A before imaging, a treatment previously shown to abrogate mitochondrial permeability increases in response to 10 μM taxol (7). Addition of 800 ng/ml taxol to cyclosporin A pretreated cells induced oscillations in 55% of all cells, which was significantly different from vehicle stimulation (Fig. 1 E) but similar to cells not treated with cyclosporin A (Table 1). Furthermore, the ability to alter the Ca2+ oscillations with complete depolarization of the mitochondrial membrane potential Ψm was tested. The addition of the mitochondrial uncoupler FCCP (1 μM) led to a fast and complete release of mitochondrial Ca2+ (first peak in Fig. 1 F), and further addition of 10 μM FCCP did not produce any additional effects on cytosolic Ca2+. Subsequent treatment with 800 ng/ml taxol still induced cytosolic Ca2+ oscillations (Fig. 1 F), which were significantly different from the response to vehicle (Table 1).

These findings were supported by direct observation of the mitochondrial membrane potential using the fluorescent potentiometric dye rhodamine 123. We observed a highly specific labeling of mitochondria, which did not change when 800 ng/ml taxol was added, indicating a stable ψm. In addition, there were no changes observed in the shape of the mitochondria, whereas 1 μM FCCP rapidly and completely depolarized the mitochondria and abolished mitochondrial labeling (Fig. 4, which is published as supporting information on the PNAS web site). An involvement of mitochondrial permeability transition in the response to low concentrations of taxol thus seems unlikely.

Taxol-Induced Ca2+ Oscillations Depend on Ca2+ Stored in the Endoplasmic Reticulum (ER) and the InsP3R.

To further dissect the mechanism of the observed taxol-induced oscillations, the Ca2+ stored in the ER was depleted with thapsigargin, an inhibitor of the sarcoplasmic-ER Ca2+ ATPase. After the initial Ca2+ release from the ER, cytosolic Ca2+ concentrations returned to baseline levels, and addition of 800 ng/ml taxol did not elicit any further Ca2+ response (Fig. 2 A). No cell produced an oscillatory response after stimulation with taxol (Table 1). Ca2+ release from the ER is thus required for the induction of Ca2+ oscillations by low concentrations of taxol.

Fig. 2.
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Fig. 2.

Taxol-induced Ca2+ oscillations depend on ER Ca2+, InsP3R, and InsP3 but not RyR. (A) Taxol-induced Ca2+ responses are abolished after depletion of ER–Ca2+ with thapsigargin. (B) Preincubation with 70 μM dantrolene decreases the response amplitude to 800 ng/ml taxol (arrow). (C and D) Preincubation with the InsP3R inhibitors xestospongin C (5 μM; black trace) or 2-APB (20 μM; gray trace) (C) and treatment with the phospholipase C inhibitor U-73122 (5 μM) (D) abolish the response to 800 ng/ml taxol (arrow). A, 10 μM ATP; T, 3 μM thapsigargin.


Ca2+ can be released from the ER through the ryanodine receptor (RyR) or InsP3R families. RyR were blocked with the cell-permeant inhibitor dantrolene. After preincubation for at least 5 min with 70 μM dantrolene, 24% of all cells produced an oscillatory response after stimulation with 800 ng/ml taxol (Table 1). This was different from cells stimulated with taxol in the absence of dantrolene and cells treated with vehicle alone, albeit the latter effect failed statistical significance because of increased variability in the response of dantrolene-treated cells. Analysis of the power spectrum density revealed a mean oscillatory frequency of 10 ± 1.1 mHz, which was not significantly different from the mean oscillatory frequency observed in taxol-stimulated cells in the absence of dantrolene. We also observed a reduction of the amplitude of the Ca2+ peaks during the oscillation in dantrolene-treated cells (Fig. 2 B). Because RyR are important for Ca2+-induced Ca2+ release (CICR) (16), it is likely that RyR participate in taxol-induced Ca2+ oscillations via CICR-mediated amplification of the signal. Because of the reduced amplitude of the Ca2+ signal, fewer cells met the criteria (Materials and Methods) we had defined for an oscillation. This observation is comparable to previous studies that showed that blockage of the RyR with dantrolene altered the shape of the response but was not required for the initiation of the response (17).

To study the role of the InsP3R in taxol-induced Ca2+ oscillations, cells were treated with the InsP3R inhibitors 2-aminoethoxydiphenylborate (2-APB) or xestospongin C. Because 100 μM 2-APB has also been shown to block the mitochondrial permeability transition pore (18), we used a concentration five times lower. Ca2+ oscillations induced by 800 ng/ml taxol were abolished in cells pretreated for at least 5 min with either 20 μM 2-APB or 5 μM xestospongin C. In some cells a small transient response was observed, but most cells did not respond at all to stimulation with taxol (Fig. 2 C and Table 1). This observation strongly supports the conclusion that the InsP3R is required for the oscillatory response of cytosolic Ca2+ to submicromolar taxol concentrations.

To determine whether taxol is able to activate the InsP3R in the absence of InsP3 or whether it modulates the response to InsP3, we blocked the formation of InsP3 with the cell-permeant phospholipase C inhibitor U-73122. Incubation for 7 min in 5 μM U-73122 abrogated the response (Fig. 2 D and Table 1). These results demonstrate that the oscillatory response to low taxol concentrations depends on phospholipase C activity, as well as activation of the InsP3R.

Screening and Identification of NCS-1 with a Biotinylated Taxol Probe.

Because taxol mainly interacts with tubulin and the observed oscillatory Ca2+ response depends on the InsP3R it was important to know whether tubulin interacts with this receptor and whether addition of 800 ng/ml taxol would cause any change in the interaction. However, no indication was found for an association of these proteins when using coimmunoprecipitation from cerebellar lysate. Only the protein directly associated with the immunoprecipitating antibody could be detected regardless of the presence of taxol (Fig. 3 A).

Fig. 3.
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Fig. 3.

Taxol binds to NCS-1, an interaction that enhances binding of NCS-1 to InsP3R. (A) Coimmunoprecipitation of β-tubulin and InsP3R from mouse cerebellar lysate. Lanes, from left to right, show mouse cerebellar lysate, beads treated with preimmune serum but no specific antibody, immunoprecipitate with anti-InsP3R1, immunoprecipitate with anti-InsP3R1 and 800 ng/ml taxol, immunoprecipitate with anti-β-tubulin, and immunoprecipitate with anti-β-tubulin and 800 ng/ml taxol. The immunoblot was probed with anti-InsP3R in Upper and anti-β-tubulin in Lower. (B) Binding analysis of 7-bio-taxol 4 with NCS-1 phage. Phage rescue titer is reported in pfu as a function of probe concentration. (C) Taxol increases the binding of NCS-1 to the InsP3R. Lanes, from left to right, show mouse cerebellar lysate, beads treated with preimmune serum, immunoprecipitate with anti-InsP3R, and immunoprecipitate with anti-InsP3R and 800 ng/ml taxol. The immunoblot was probed with anti-InsP3R1 in Upper and anti-NCS-1 in Lower. (D) Cells transiently transfected with a vector expressing NCS-1 shRNA as well as GFP showed a significant reduction (≈80%) of the immunosignal compared with cells expressing scrambled shRNA. (E) Oscillations induced by 800 ng/ml taxol (arrow) were abrogated in NCS-1 knockdown cells (red trace) but unaffected in cells expressing scrambled shRNA (black trace). (F) Oscillations induced by 0.75 μM ATP were unaffected by NCS-1 knockdown (red trace) compared with scrambled shRNA-expressing cells (black trace). T, 3 μM thapsigargin.


Because known taxol interaction partners could not explain our observations and several taxol-specific side effects involve the peripheral nervous system (6) we screened a human brain T7 cDNA phage display library for taxol binding using C-7-biotinylated-taxol (7-bio-taxol) and a modified display cloning procedure (11). Each selection round in display cloning involved three distinct steps: (i) binding of the phage to the solid support, (ii) washing away unbound phage, and (iii) eluting bound phage particles, which were used for the next round of selection. After the first round of affinity selection, 16 clones were selected and analyzed by PCR amplification of the insert region and DNA sequencing. None of the 16 clones contained an expressible gene. After the second round of affinity selection, 3 of 16 clones were found to encode the full ORF of NCS-1 (Fig. 5A, which is published as supporting information on the PNAS web site) (19). The coding sequence was in-frame with the coding sequence of cp10, the T7 coat protein, as expected for a properly displayed protein. After the third round of selection 8 of 16 clones contained NCS-1, with four clones not having inserts at all. The predominance of NCS-1 in the rescued clones strongly suggested that NCS-1 was being affinity-amplified by 7-bio-taxol. In addition, the absence of additional ORFs, as seen in other selections (20) and the appearance of phage clones not containing any gene inserts, suggested that NCS-1 was the primary binding protein for taxol in this cDNA library. When using biotinylated phorbol ester and identical selection procedures, none of the isolated clones were NCS-1, even after five rounds of selection.

To validate the affinity of 7-bio-taxol for the NCS-1 protein, an on-phage concentration-dependent binding study was performed. The phage solutions were titered and plotted as a function of probe concentration for each well. Nonlinear regression analysis of the data gave an EC50 value of 728 ± 44 ng/ml (557 ± 34 nM) (Fig. 3 B). This result is intriguing because the concentration is within the range observed in patients (9) and, as outlined above, sufficient to induce Ca2+ oscillations. Crystal structure analysis of human NCS-1 has shown that, upon treatment with Ca2+, three of its four EF motifs bind the metal, causing a large conformational shift in the C-terminal portion of the protein and exposing a large hydrophobic cleft, which is postulated to serve as a protein docking site that forms in response to cellular Ca2+ flux (19). It is possible that this region of the protein also serves as the binding site for the taxol probe molecule. Given the large size of this hydrophobic pocket, we evaluated four other biotinylated natural products, as well as a molecule that matches the linker portion of 7-bio-taxol for their capability to bind NCS-1 phage. None of the additional probe molecules, bio-FK506, bio-phorbol ester, bioflavokavin A, biobengamide, and the biotin-LC-LC linker, exhibited any appreciable binding to NCS-1 phage (Fig. 5B). Together these results indicate that bio-taxol's affinity for the NCS-1-expressing phage clone is specific and not due to nonspecific hydrophobic interaction.

NCS-1 Knockdown Abrogates Taxol-Induced Ca2+ Oscillations.

Because taxol binds to NCS-1 but not to the InsP3R and NCS-1 is capable of positively modulating InsP3-mediated Ca2+ release (13), we hypothesized that the observed oscillatory response to submicromolar concentrations of taxol might be due to an alteration of the NCS-1–InsP3R interaction. To study whether the binding of NCS-1 to the InsP3R is altered in the presence of 800 ng/ml taxol, NCS-1 was coimmunoprecipitated with InsP3R from cerebellar lysate. The amount of NCS-1 bound to the InsP3R was increased in the presence of taxol (Fig. 3 C).

To further establish the importance of the NCS-1–InsP3R interaction for the observed effects, NCS-1 was knocked down by transient transfection with a vector coexpressing anti NCS-1 shRNA and GFP. The coexpression with GFP allowed identification of those cells expressing the NCS-1 shRNA, which would contain less NCS-1 protein. Ca2+ transients were monitored in cells by using the red fluorescent dye Rhod-2/AM. Expression of NCS-1 shRNA resulted in a reduction in the immunosignal for NCS-1 by ≈80% (n = 3 independent experiments; P < 0.001) (Fig. 3 D).

Interestingly, the oscillatory Ca2+ response to 800 ng/ml taxol was abrogated in NCS-1 knockdown cells compared with cells transfected with a vector expressing a scrambled shRNA sequence that does not target any known gene (Table 1). There were no responses to taxol stimulation in 72% of NCS-1 knockdown cells compared with 4% (P < 0.001) of cells transfected with scrambled shRNA (Fig. 3 E). The responses of the NCS-1 knockdown cells were not significantly different from vehicle treatment (Table 1). These results indicate that NCS-1 is causally involved in the Ca2+ response to low taxol concentrations.

Because NCS-1 positively modulates the InsP3R-mediated Ca2+ release (13), we tested whether NCS-1 knockdown cells were still able to oscillate in response to stimulation with low concentrations of ATP, a robust activator of InsP3-dependent Ca2+ oscillations. Induction of Ca2+ oscillations was not affected in NCS-1 knockdown cells with 42 ± 7% [70/4] of all cells oscillating upon stimulation with 0.75 μM ATP compared with 38 ± 13% [64/4] (not significant) of cells transfected with scrambled shRNA (Fig. 3 F). This result supports the suggestion that the observed abrogation of taxol-induced Ca2+ oscillations in NCS-1 knockdown cells is taxol-specific and not a nonspecific side effect of the NCS-1 knockdown.

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Discussion

The aim of this study was to investigate the effects of submicromolar concentrations of taxol, as found in patients undergoing chemotherapy for solid tumors (9, 14), on the Ca2+ homeostasis of a neuronal cell line. We found a rapid induction of cytosolic Ca2+ oscillations, which, at least initially, did not depend on Ca2+ fluxes from the extracellular space or on the release of mitochondrial Ca2+. A further dissection of the molecular basis of this signal revealed a dependence on Ca2+ release from the ER and, more specifically, the InsP3R. Release of Ca2+ through the RyR does not seem to be necessary for the initiation of the Ca2+ transient under these conditions, but activation of the RyR is used for amplification of the Ca2+ signal. Because known taxol binding proteins could not explain this effect, we used a human brain cDNA phage display library and affinity chromatography with a biotinylated taxol probe to screen for new interaction partners of taxol. By using this procedure, a phage clone that encodes the NCS-1 protein was isolated. Subsequent binding analyses confirmed the affinity of taxol for this protein and established both probe- and Ca2+-dependent binding characteristics. Intriguingly, the concentration range for the affinity of bio-taxol for NCS-1 was well within the concentration range inducing Ca2+ oscillations.

Recently we demonstrated that NCS-1 enhances InsP3R activity both at the single-channel level and in intact cells (13). This led us to hypothesize that the observed oscillatory response to submicromolar concentrations of taxol is due to a NCS-1-mediated increase in InsP3R activity, which should be abrogated in cells where NCS-1 is knocked down. Indeed, we found an increased binding of NCS-1 to the InsP3R in the presence of taxol, and when NCS-1 was knocked down the Ca2+ response was not significantly different from vehicle treatment. The latter effect appears to be taxol-specific, because NCS-1 knockdown cells oscillated to the same extent as cells with normal NCS-1 levels after stimulation with low concentrations of ATP. The key findings of this study have implications for various taxol therapy-related side effects, discussed below and outlined in a model (Fig. 6, which is published as supporting information on the PNAS web site).

One side effect, which almost led to the discontinuation of clinical trials with taxol, is the high incidence of severe hypersensitivity reactions (21). This problem could be addressed with a stringent pretreatment of all patients with corticosteroids as well as H1 and H2 antagonists (21). However, even with pretreatment, between 1% and 3% of all patients develop major hypersensitivity reactions (6). Initially it was thought that the vehicle cremophore EL was responsible for these hypersensitivity reactions (reviewed in ref. 14), but subsequent elegant in vivo studies linked this phenomenon to sensory nerve peptides such as Substance P and calcitonin gene-related peptide (CGRP) (22). By using these newer findings it was suggested that release of the peptide mediators would cause mast cell activation with histamine release. Notably, NCS-1 is highly expressed by dorsal root ganglion cells and has been found to be colocalized with CGRP in peripheral nerve terminals innervating blood vessels (23). In light of our findings, it seems likely that binding of taxol to NCS-1 in peripheral nerves could facilitate the release of neuropeptides in a Ca2+-dependent fashion, which can then trigger a hypersensitivity reaction.

Another side effect with a high incidence is cardiac arrhythmia (24). This phenomenon does not seem to be linked to cytotoxicity and could be reproduced in an in vitro preparation (25). Although the InsP3R is not playing a dominant role in excitation–contraction coupling in cardiomyocytes, several studies suggest a role for the InsP3R in cardiac arrythmogenesis (26). Because NCS-1 is also expressed in the heart (27), one direct prediction from our observations, that taxol positively modulates the InsP3R in a NCS-1-dependent manner, would be a positive inotropic effect (26); this has been observed in taxol-treated papillary muscles (25).

NCS-1 is also highly expressed in neuronal tissues. Because taxol does not seem to cross an intact blood–brain barrier (28), neuronal side effects should occur in the peripheral nervous system. In fact, peripheral neuropathy (PNP) is another frequent major dose-dependent and therapy-limiting side effect of taxol chemotherapy that is still poorly understood (reviewed in ref. 29). In the context of the present study, it is interesting to note that inhibition of the Ca2+-activated calpain proteases has a protective effect against taxol-induced sensory neuropathy in vivo and taxol causes activation of both calpains and caspases in PC12 cells (30). Furthermore, the Ca2+-permeable nonselective cation channel transient receptor potential vanilloid 4 (TRPV4), a receptor located in the plasma membrane, has been shown to be essential in taxol-induced PNP in rats (31). This last observation is intriguing because TRPV4 currents were shown to be potentiated by increases in intracellular Ca2+ (32). It thus seems likely that cytosolic Ca2+ oscillations in dorsal root ganglia neurons are induced by taxol binding to NCS-1 and subsequent positive modulation of the InsP3R. These effects, in turn, are potentiated in a TRP-dependent manner and lead to the activation of Ca2+ activated proteases with the result of cell malfunction and death, resulting in PNP. This model offers an explanation for the apparent beneficial effects of Ca2+ channel blockers in the treatment of PNP (29).

Taxol is also frequently used as a pharmacological tool in cell biology, where it is used to arrest cells in the G2 phase, study microtubule function, and visualize the microtubule cytoskeleton with fluorophore-conjugated derivatives. In the context of the present finding that taxol also affects intracellular Ca2+ signaling, care should be given when interpreting the results obtained with taxol as a microtubule-modifying drug.

In summary, our observations (that taxol has a new binding partner, NCS-1, and that binding to NCS-1 leads to initiation of cytosolic Ca2+ oscillations) suggest that taxol's effects when used as a research tool will be more complex than originally expected and that our model (Fig. 6) may help to devise new strategies for the management of side effects induced by taxol therapy.

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Materials and Methods

Plasmids.

The GeneClip U1 Hairpin Cloning Systems (Promega, Madison, WI) was used as vector for expression of NCS-1 or scrambled shRNA. The shRNA template for NCS-1 was GGCTTCCAGAAGATCTACA, and as scrambled sequence for controls we used GGCTTCGTGAAGGTCTATA.

Cell Cultures and Transfection.

The human neuroblastoma cell line SH-SY5Y (American Type Culture Collection, Manassas, VA) was cultured and transfected as described previously (33).

Ca2+ Imaging.

Cells were incubated (30 min at 37°C in 5% CO2) in Hepes buffer containing either 5 μM Fluo-4/AM or 6 μM Rhod-2/AM (Molecular Probes, Invitrogen, Carlsbad, CA) together with 0.1% Pluronic F-127 (Molecular Probes, Invitrogen). The Hepes medium contained 130 mM NaCl, 4.7 mM KCl, 1 mM MgSO4, 1.2 mM KH2PO4, 1.3 mM CaCl2, 20 mM Hepes, and 5 mM glucose (pH 7.4). Coverslips were mounted in a chamber (Warner Instruments, Hamden, CT) and transferred to a Zeiss LSM 510 META scanning laser confocal microscope equipped with a C-Apochromat ×40/1.2 water immersion objective (Zeiss, Thornwood, NY). Images were acquired at 0.33 Hz. All drugs were bath-applied. To identify cells expressing shRNA with GFP, transfected cells were examined by using fluorescence excitation at 488 nm in a multitrack configuration to minimize crosstalk. Whole-cell fluorescence was measured by defining each cell as one region of interest. A given cell was considered to oscillate when at least three Ca2+ transients (deflections from ≥20% from baseline) were recorded over the time monitored, usually 10 min. Cells that did not respond to stimulation and to control stimulation with either 10 μM ATP (A) or 3 μM thapsigargin (T), as indicated in Results, were excluded from evaluation. Inhibitors were added after the dye incubation; times and concentrations are indicated in Results. Ca2+-induced fluorescence intensity ratio F/F 0 was plotted as a function of time with F 0 as an average of the first five points of the baseline. To perform power spectrum analysis, we used an algorithm written in MATLAB as described previously (15).

Statistical Analysis.

Data are expressed as mean ± SEM or as representative traces. [n/N] describes the number of cells studied (n) in N independent cultures. Statistical analysis of the differences between multiple groups was performed by using a one-way ANOVA (Dunnett multiple-comparisons test) (Instat; GraphPad, San Diego, CA) for two groups using t test (SigmaPlot, Systat, Richmond, CA); P < 0.05 was considered statistically significant.

Immunoprecipitation and Western Blot Analysis.

Lysate preparation, immunoprecipitation, and immunoblotting were performed as described previously (13). Antibodies used were as follows: anti-NCS-1 (Santa Cruz Biotechnology, Santa Cruz, CA), anti-β-actin (Abcam, Cambridge, MA), anti-β-tubulin (Covance, Berkeley, CA), and anti-InsP3R1 (17). For immunoprecipitation, cerebellar lysate was incubated with antibody in the presence of 50 μM free Ca2+ and 800 ng/ml taxol as indicated in Results. NCS-1 knockdown blots were quantified by scanning densitometry by using UN-SCAN-IT (Silk Scientific, Orem, UT) normalizing NCS-1 expression to the β-actin loading control.

Display Cloning.

Human brain polyA+ mRNA (Clontech, Mountain View, CA) was used to create a cDNA phage display library with the OrientExpress cDNA library synthesis kit (Novagen, La Jolla, CA). Phage lysate generation for T7 cDNA libraries and individual T7 phage clones were prepared by infection into log phase BLT5403 Escherichia coli. The oligonucleotide sequencing primers T7Forward (5′-TCTTCGCCCAGAAGCTGCAG) and T7Reverse (5′-CCTCCTTTCAGCAAAAAACCCC) were used for both PCR amplification and DNA sequence analysis of the T7 phage-displayed inserts. C7-bio-taxol agarose columns were prepared by incubating a slurry of avidin agarose resin from Pierce (Rockford, IL) in PBS with C7-bio-taxol. The column was extensively washed with wash buffer (50 mM Tris, 150 mM NaCl, 0.5 mM CaCl2, and 1 mM MgSO4), after which an aliquot of the cleared human brain T7 cDNA phage display lysate was added to the resin. Nonspecifically bound phage was removed by washing with wash buffer. Bound phage were eluted with 1% SDS in PBS, and the eluent was diluted with LB media (1:1). Phage rescue titer was found to be 26 × 106 pfu/ml after the first round of selection, 54 × 106 pfu/ml after the second round, and 3,000 × 106 pfu/ml after the third round. Random selection of phage plaques and sequencing yielded multiple copies of an identical clone, encoding NCS-1 protein. The clone TT3.5 was selected for use in all subsequent studies.

For probe-dependent NCS-1 phage binding studies, serial dilutions of each biotinylated probe were incubated with a NeutrAvidin-coated microtiter plate (Pierce), and subsequently a biotin block (1 mM) was performed to reduce background binding. An aliquot of the NCS-1 phage lysate was added to each well and incubated overnight at 4°C. Nonbinding phage were removed by washing, and bound phage were eluted by treatment with 1% SDS in TBS. The eluate was titered by serial dilution and dropping triplicates onto bacterial plates. The resulting plaques were counted and plotted as a function of incubated biotinylated probe. Information about the additional methods used for Figs. 4–6 can be found in Supporting Experimental Procedures, which is published as supporting information on the PNAS web site.

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Acknowledgments

We thank Manuel Estrada, Per Uhlen, Anurag Varshney, Sven-Eric Jordt, Brenda DeGray, Felix Heidrich, and Victor K. Chung for invaluable advice regarding the design of the experiments and thoughtful discussions and comments on the manuscript. We also thank M. Venkata Rami Reddy (University of California, La Jolla, CA) for the synthesis of C7-bio-taxol and Andreas Jeromin (University of Texas, Austin, TX) and Jolanta Vidugiriene (Promega, Madison, WI) for shRNA reagents. This work was supported by National Institutes of Health Grants GM63496 (to B.E.E.) and GM59673 (to D.J.A.), the Patterson Research Trust (B.E.E.), National Center for Complementary and Alternative Medicine Small Business Innovation Research Phase I Grant R43 AT0 0324-01 (to Ancile Pharmaceuticals), and a German National Merit Foundation scholarship (to W.B.). D.J.A. was a Fellow of the Alfred P. Sloan Foundation.

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Footnotes

  • §To whom correspondence should be addressed at:
    Department of Pharmacology, Yale University School of Medicine, 333 Cedar Street, New Haven, CT 06520-8066.
    E-mail: barbara.ehrlich{at}yale.edu

  • Author contributions: W.B., D.J.A., and B.E.E. designed research; W.B., U.S., M.B.L., K.M.M., and D.G.J., performed research; W.B., U.S., M.B.L., and K.M.M. analyzed data; and W.B., B.E.E., and D.J.A. wrote the paper.

  • The authors declare no conflict of interest.

  • This article is a PNAS direct submission.

  • Abbreviations:
    ER,
    endoplasmic reticulum;
    InsP3R,
    inositol 1,4,5-trisphosphate receptor;
    NCS-1,
    neuronal Ca2+ sensor 1;
    RyR,
    ryanodine receptor;
    2-APB,
    2-aminoethoxydiphenylborate;
    shRNA,
    short hairpin RNA;
    PNP,
    peripheral neuropathy.
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