p53 is associated with cellular microtubules and is transported to the nucleus by dynein.

Here we show that p53 protein is physically associated with tubulin in vivo and in vitro, and that it localizes to cellular microtubules. Treatment with vincristine or paclitaxel before DNA-damage or before leptomycin B treatment reduces nuclear accumulation of p53 and expression of mdm2 and p21. Overexpression of dynamitin or microinjection of anti-dynein antibody before DNA damage abrogates nuclear accumulation of p53. Our results indicate that transport of p53 along microtubules is dynein-dependent. The first 25 amino acids of p53 contain the residues that are essential for binding to microtubules. We propose that functional microtubules and the dynein motor protein participate in transport of p53 and facilitate its accumulation in the nucleus after DNA damage.

Regulation of apoptosis in myeloid cells by interferon consensus sequence-binding protein.

Mice with a null mutation of the gene encoding interferon consensus sequence-binding protein (ICSBP) develop a disease with marked expansion of granulocytes and macrophages that frequently progresses to a fatal blast crisis, thus resembling human chronic myelogenous leukemia (CML). One important feature of CML is decreased responsiveness of myeloid cells to apoptotic stimuli. Here we show that myeloid cells from mice deficient in ICSBP exhibit reduced spontaneous apoptosis and a significant decrease in sensitivity to apoptosis induced by DNA damage. In contrast, apoptosis in thymocytes from ICSBP-deficient mice is unaffected. We also show that overexpression of ICSBP in the human U937 monocytic cell line enhances the rate of spontaneous apoptosis and the sensitivity to apoptosis induced by etoposide, lipopolysaccharide plus ATP, or rapamycin. Programmed cell death induced by etoposide was specifically blocked by peptides inhibitory for the caspase-1 or caspase-3 subfamilies of caspases. Studies of proapoptotic genes showed that cells overexpressing ICSBP have enhanced expression of caspase-3 precursor protein. In addition, analyses of antiapoptotic genes showed that overexpression of ICSBP results in decreased expression of Bcl-X(L). These data suggest that ICSBP modulates survival of myeloid cells by regulating expression of apoptosis-related genes.

Brain-derived neurotrophic factor protects neuroblastoma cells from vinblastine toxicity.

Brain-derived neurotrophic factor (BDNF) and its receptors are necessary for the survival and development of many neuronal cells. Because BDNF and TrkB are expressed in many poor-prognosis neuroblastoma (NB) tumors, we evaluated the role of BDNF in affecting sensitivity to chemotherapeutic agents. We investigated the effects of activation of the BDNF-TrkB signal transduction pathway in two NB cell lines, 15N and SY5Y. 15N cells lack the high-affinity receptor p145TrkB and express BDNF; 15N cells were used along with 15N-TrkB cells, a subline transfected with a TrkB expression vector. In cytotoxicity assays, 15N-TrkB cells were consistently 1.4-2 fold more resistant to vinblastine than 15N cells. Drug accumulation assays showed a 50% reduction in[3H]vinblastine accumulation in 15N-TrkB cells compared with control 15N cells. Addition of 30 ng/ml BDNF resulted in a reduction to 46% of control in 15N cells and a reduction to 28% of control in 15N-TrkB cells. SY5Y cells were chosen as a second model because they lack both endogenous BDNF and TrkB expression. p145TrkB expression is induced by 1 nM retinoic acid. Vinblastine accumulation was not significantly affected by 1 nM retinoic acid in SY5Y cells. Addition of 30 ng/ml BDNF decreased [3H]vinblastine accumulation to 58% of control in SY5Y cells and decreased [3H]vinblastine accumulation to 62% of control in TrkB-expressing SY5Y cells. Although an increase in BDNF expression in seen in multidrug-resistant sublines of SY5Y and BE(2)-C NB cells, the protective effect of BDNF in vinblastine toxicity may be unrelated to mdr-1, because the activity of other agents transported by P-glycoprotein was not affected. There was no increase in mdr-1 expression in 1 nM RA SY5Y cells and 15N-TrkB cells, as assessed by Northern blot analysis. In addition to the effects of BDNF on vinblastine cytotoxicity and accumulation, there was an inhibition in the ability of vinblastine to depolymerize tubulin in BDNF-treated cells. Thus, BDNF and TrkB may partially rescue NB cells from vinblastine toxicity and thereby may contribute to a more chemoresistant phenotype.

Raf-1/bcl-2 phosphorylation: a step from microtubule damage to cell death.

Recent studies have shown that paclitaxel leads to activation of Raf-1 kinase and have suggested that this activation is essential for bcl-2 phosphorylation and apoptosis. In the present study, we demonstrate that, in addition to paclitaxel, other agents that interact with tubulin and microtubules also induce Raf-1/bcl-2 phosphorylation, whereas DNA-damaging drugs, antimetabolites, and alkylating agents do not. Activation of Raf-1 kinase by paclitaxel is linked to tubulin polymerization; the effect is blunted in paclitaxel-resistant cells, the tubulin of which does not polymerize following the addition of paclitaxel. In contrast, vincristine and vinblastine, drugs to which the paclitaxel-resistant cells retain sensitivity were able to bring about Raf-1 phosphorylation. The requirement for disruption of microtubules in this signaling cascade was strengthened further using paclitaxel analogues by demonstrating a correlation between tubulin polymerization, Raf-1/bcl-2 phosphorylation, and cytotoxicity. Inhibition of RNA or protein synthesis prevents Raf-1 activation and bcl-2 phosphorylation, suggesting that an intermediate protein(s) acts upstream of Raf-1 in this microtubule damage-activating pathway. A model is proposed that envisions a pathway of Raf-1 activation and bcl-2 phosphorylation following disruption of microtubular architecture, serving a role similar to p53 induction following DNA damage.

Low concentrations of paclitaxel induce cell type-dependent p53, p21 and G1/G2 arrest instead of mitotic arrest: molecular determinants of paclitaxel-induced cytotoxicity.

Paclitaxel (PTX), a microtubule-active agent, blocks cell proliferation by inhibiting mitotic progression leading to mitotic and postmitotic arrest and cell death. Here we demonstrate for the first time that very low concentrations of PTX (3-6 nM) can completely inhibit cell proliferation without arresting cells at mitosis. At these low concentrations that are insufficient to inhibit mitotic progression, PTX induced both p53 and p21 causing G1 and G2 arrest in A549. In contrast, low PTX concentrations failed to induce G1 and G2 arrest in A549/E6 cells, that do not express p53. Furthermore, we observed that the levels of p53 and p21 induced by adriamycin and by low concentrations of PTX in A549 cells were comparable. This observation led us to conclude that low concentrations of PTX can induce p53 and p21 sufficiently to cause G1 and G2. Many other cell lines, including HCT116 cells, do not readily upregulate p53 in response to PTX, and therefore undergo exclusively mitotic and postmitotic arrest after PTX treatment. At low concentrations that do not cause mitotic arrest, PTX did not significantly inhibit proliferation of these cells. In HCT116 cells, loss of p53 (HCT/p53(-/-)) or p21 (HCT/p21(-/-)) affects both Bax and Bcl-2 expression. In cells lacking p53, levels of Bax and p21 were decreased. In cells lacking p21, levels of wt p53 were highly increased to compensate for the loss of p21. This in turn results in upregulation of Bax and downregulation of Bcl-2 resulting in an increase of the apoptotic Bax/Bcl2 ratio consistent with increased sensitivity of these cells to apoptotic stimuli. High levels of p53 and Bax/Bcl-2 ratio can also explain why loss of p21 is rarely found in human cancer.

Paclitaxel selects for mutant or pseudo-null p53 in drug resistance associated with tubulin mutations in human cancer.

The efficacy of anticancer therapy is limited by the development of drug resistance. While the role of p53 in the intrinsic sensitivity of human cancer cells to paclitaxel (PTX) remains controversial, its role in acquired paclitaxel resistance has never been addressed. In this study we examined the p53 status of three paclitaxel selected human ovarian carcinoma sublines, resistant to paclitaxel due to acquired beta-tubulin mutations which impair paclitaxel's interaction with tubulin. In contrast to parental cells which have wt p53, in all PTX-resistant sublines p53 was functionally inactive. Two of the resistant sublines expressed high levels of transcriptionally inactive p53 protein, each with a distinct point mutation in codons 236 and 239 of the DNA binding domain. The third subline presented a novel p53 pseudo-null phenotype as a result of markedly decreased wt p53 mRNA expression. Introduction of ectopic wt p53 had no effect on PTX sensitivity in both parental and resistant cells, while it induced p21WAF1/CIP1, demonstrating an intact p53 pathway. While PTX resistance is primarily conferred by the tubulin mutations, the loss of functional p53 observed in all clones, suggests that this loss may facilitate the development of resistance potentially by providing a clonal advantage which promotes the isolation of paclitaxel resistant cells.

Chemical synthesis and biological properties of pyridine epothilones.

BACKGROUND: Numerous analogs of the antitumor agents epothilones A and B have been synthesized in search of better pharmacological profiles. Insights into the structure-activity relationships within the epothilone family are still needed and more potent and selective analogs of these compounds are in demand, both as biological tools and as chemotherapeutic agents, especially against drug-resistant tumors. RESULTS: A series of pyridine epothilone B analogs were designed, synthesized and screened. The synthesized compounds exhibited varying degrees of tubulin polymerization and cytotoxicity properties against a number of human cancer cell lines depending on the location of the nitrogen atom and the methyl substituent within the pyridine nucleus. CONCLUSIONS: The biological screening results in this study established the importance of the nitrogen atom at the ortho position as well as the beneficial effect of a methyl substituent at the 4- or 5-position of the pyridine ring. Two pyridine epothilone B analogs (i.e. compounds 3 and 4) possessing higher potencies against drug-resistant tumor cells than epothilone B, the most powerful of the naturally occurring epothilones, were identified.

Expression of beta-tubulin isotypes in human ovarian carcinoma xenografts and in a sub-panel of human cancer cell lines from the NCI-Anticancer Drug Screen: correlation with sensitivity to microtubule active agents.

Paclitaxel resistance has been associated with overexpression of P-glycoprotein and alterations involving tubulin. To investigate the clinical relevance of these in vitro resistance mechanisms, we established 12 human ovarian carcinoma xenografts, using samples from patients before the start of therapy or after paclitaxel treatment. These xenografts showed a wide range of sensitivity to paclitaxel, and in 4 of them, very low levels of multidrug resistance-1 expression were detected. Using quantitative PCR and human specific primers, the expression of five beta-tubulin isotypes was determined. HM40 was the predominant, accounting for 84.7-98.7% of all tubulin; expression of the other four isotypes (Hbeta9, Hbeta4, H5beta, and Hbeta2) was also detected but at lower levels. No correlation could be demonstrated between isotype expression and paclitaxel sensitivity in these 12 xenografts. A similar pattern of beta-tubulin isotype expression was observed in a subset of cell lines from the National Cancer Institute-Anticancer Drug Screen. In these cell lines, however, a significant correlation between increased expression of Hbeta4 isotype and resistance to paclitaxel was found. Taken together, these results suggest that altered expression of specific beta-tubulin isotypes may not play a significant role in paclitaxel sensitivity in vivo and argue against a possible significance in a clinical setting.

Cytotoxic chemotherapy in the treatment of advanced renal cell carcinoma in the era of targeted therapy.

BACKGROUND: Renal cell carcinoma (RCC) is a heterogeneous disease with regards to histology, progression, and response to treatment. Cytotoxic chemotherapy has been extensively studied in metastatic RCC (mRCC). Responses in most studies are modest and the mechanisms of resistance remain poorly understood. Targeted therapies have significantly improved outcomes in mRCC; however, most patients eventually relapse and die of their disease. Early clinical data suggest that combinations of chemotherapy and targeted agents are clinically active and are well tolerated. METHODS: We reviewed the available literature for published clinical trials incorporating traditional chemotherapeutic agents in the treatment of mRCC. These papers were identified through a Medline search and were included if they employed at least one chemotherapeutic agent in the treatment of mRCC. The literature was also reviewed for information regarding mechanisms of chemotherapy resistance. RESULTS: The data regarding the use of cytotoxic chemotherapy in mRCC consist of small, non-randomized phase I and II studies. The major response proportions with single agent chemotherapies are low but combination regimens either with other cytotoxic agents, cytokines, or targeted agents have demonstrated moderate activity. Disparate trial designs and lack of head to head clinical trials make it difficult to compare the efficacy of chemotherapy with that of immunotherapy or targeted agents. Chemotherapy is particularly useful in patients with collecting duct histology and predominantly sarcomatoid differentiation. Chemotherapy resistance may be mediated by overexpression of p-glycoprotein efflux pumps and the dysregulation of the microtubule-hypoxia inducible factor signaling axis. CONCLUSIONS: The role of cytotoxic chemotherapy in the treatment for clear cell RCC remains poorly defined. Cytotoxic chemotherapy is considered a standard of care in patients with mRCC with predominantly sarcomatoid differentiation and collecting duct RCC variants (Motzer et al., 2014). Early trials combining chemotherapy with targeted therapies are generally well tolerated and show clinical activity. A better understanding of the biology of aggressive subsets of RCC and mechanisms of resistance will help elucidate the role of cytotoxic agents in the current treatment paradigm of RCC.

ß-1 tubulin R307H SNP alters microtubule dynamics and affects severity of a hereditary thrombocytopenia.

BACKGROUND: Single nucleotide polymorphisms (SNPs) in platelet-associated genes partly explain inherent variability in platelet counts. Patients with monoallelic Bernard Soulier syndrome due to the Bolzano mutation (GPIBA A156V) have variable platelet counts despite a common mutation for unknown reasons. OBJECTIVES: We investigated the effect of the most common SNP (R307H) in the hematopoietic-specific tubulin isotype ß-1 in these Bernard Soulier patients and potential microtubule-based mechanisms of worsened thrombocytopenia. PATIENTS/METHODS: Ninety-four monoallelic Bolzano mutation patients were evaluated for the R307H ß-1 SNP and had platelet counts measured by three methods; the Q43P SNP was also evaluated. To investigate possible mechanisms underlying this association, we used molecular modeling of ß-1 tubulin with and without the R307H SNP. We transfected SNP or non-SNP ß-1 tubulin into MCF-7 and CMK cell lines and measured microtubule regrowth after nocodazole-induced depolymerization. RESULTS: We found that patients with at least one R307H SNP allele had significantly worse thrombocytopenia; manual platelet counting revealed a median platelet count of 124 in non-SNP patients and 76 in SNP patients (both ×10(9)  L(-1) ; P < 0.01). The Q43P SNP had no significant association with platelet count. Molecular modeling suggested a structural relationship between the R307H SNP and microtubule stability via alterations in the M-loop of ß tubulin; in vitro microtubule recovery assays revealed that cells transfected with R307H SNP ß-1 had significantly impaired microtubule recovery. CONCLUSIONS: Our data show that the R307H SNP is significantly associated with the degree of thrombocytopenia in congenital and acquired platelet disorders, and may affect platelets by altering microtubule behavior.

Effects of p53-expressing adenovirus on the chemosensitivity and differentiation of anaplastic thyroid cancer cells.

We investigated the p53 status and the ability of exogenous wildtype (wt) p53 to affect chemosensitivity in three anaplastic thyroid carcinoma cell lines (BHT-101, SW-1736, and KAT-4). All three cell lines had nonfunctional p53. Treatment with mitomycin C or adriamycin did not result in accumulation of p53 or induction of p21WAF1/CIP1 or Mdm-2 and did not cause Rb dephosphorylation. BHT-101 and KAT-4 cells had mutant p53. SW-1736 cells were functionally mutant because of marked down-regulation of wt p53 messenger ribonucleic acid, representing a novel mechanism of p53 dysfunction. Infection with a p53-expressing adenovirus (Ad-p53) induced high levels of p21 and Mdm-2 proteins. In BHT-101 cells, induction of p21 and Mdm-2 was evident 10 h after infection. In KAT-4 cells, induction of p21 and Mdm-2 was observed 1 day after infection, and continued to increase over the ensuing 24 h. SW-1736 cells demonstrated intermediate kinetics. Sensitivity to the cytotoxic effect of Ad-p53 paralleled the kinetics of p21/Mdm-2 induction. BHT-101 cells were most sensitive to killing by Ad-p53, with an IC50 of less than 2 multiplicity of infection; SW-1736 cells were intermediate in sensitivity; KAT-4 cells were resistant. All three cell lines became more sensitive to adriamycin after wt p53 expression, with a 10-fold decrease in IC50 values. The latter observation may make a combination of wt p53 and chemotherapeutic drugs an attractive modality for treating anaplastic thyroid cancer.

Synthesis and biological evaluation of 2-acyl analogues of paclitaxel (Taxol).

The anticancer drug paclitaxel (Taxol) has been converted to a large number of 2-debenzoyl-2-aroyl derivatives by three different methods. The bioactivities of the resulting analogues were determined in both tubulin polymerization and cytotoxicity assays, and several analogues with enhanced activity as compared with paclitaxel were discovered. Correlation of cytotoxicity in three cell lines with tubulin polymerization activity showed reasonable agreement. Among the cell lines examined, the closest correlation with antitubulin activity was observed with a human ovarian carcinoma cell line.

Accompanying protein alterations in malignant cells with a microtubule-polymerizing drug-resistance phenotype and a primary resistance mechanism.

Microtubules (MTs) are cytoskeletal components whose structural integrity is mandatory for the execution of many basic cell functions. Utilizing parental and drug-resistant ovarian carcinoma cell lines that have acquired point mutations in beta-tubulin and p53, we studied the level of expression and modification of proteins involved in apoptosis and MT integrity. Extending previous results, we demonstrated phosphorylation of pro-survival Bcl-x(L) in an epothilone-A resistant cell line, correlating it with drug sensitivity to tubulin-active compounds. Furthermore, Mcl-1 protein turned over more rapidly following exposure to tubulin-modifying agents, the stability of Mcl-1 protein paralleling the drug sensitivity profile of the paclitaxel or epothilone-A resistant cell lines. The observed decreases in Mcl-1 were not a consequence of G(2)M arrest, as determined by flow cytometry analysis, which showed prominent levels of Mcl-1 in the absence of any drug treatment in populations enriched in mitotic cells. We also observed that a paclitaxel-resistant cell line expressed Bax at a much lower level than the sensitive parental line [A2780(1A9)], consistent with its mutant p53 status. MT-associated protein-4 (MAP4), whose phosphorylation during specific phases of the cell cycle reduces its MT-polymerizing and -stabilizing capabilities, was phosphorylated in response to drug challenge without a change in expression. Phosphorylation of MAP4 correlated with sensitivity to tubulin-binding drugs and with a dissociation from MTs. We propose that the tubulin mutations, which result in a compromised paclitaxel:tubulin or epothilone:tubulin interaction and paclitaxel or epothilone resistance, indirectly inhibit downstream events that lead to cell death, and this, in turn, may contribute to the drug-resistance phenotype

Tubulin from paclitaxel-resistant cells as a probe for novel antimicrotubule agents.

PURPOSE: Treatment with paclitaxel (PTX) can lead to the appearance of drug resistance with accompanying changes in tubulin. The purpose of this study was to develop an assay for microtubule-active agents that are able to circumvent changes in tubulin that result in acquired resistance to paclitaxel. METHODS: The assay measured the promotion of microtubule polymerization when target agents were added to solutions containing tubulin purified from cultured cells. Tubulin was prepared from PTX-sensitive 1A9 ovarian carcinoma cells and from a PTX-resistant clone. Polymerization was monitored spectrophotometrically and validated by electron microscopy. RESULTS: Exposure of tubulin isolated from PTX-sensitive 1A9 ovarian carcinoma cells to substoichiometric PTX resulted in polymerization equivalent to that observed with brain tubulin. In contrast, tubulin from a PTX-resistant 1A9 clone failed to polymerize under identical conditions. If a C-2-modified analog of PTX (2-debenzoyl-2-(m-azidobenzoyl)paclitaxel) was substituted for PTX in the same experiment, the tubulins from both sensitive and resistant cells polymerized as well as brain tubulin. As predicted from these results, the PTX analog was nearly as cytotoxic to the PTX-resistant cells as it was to the parental cells: the relative resistance of the resistant cells compared to the parental is only 3-5-fold for the PTX analog versus 25-30-fold for PTX. CONCLUSION: Polymerization of purified tubulin from the paclitaxel-resistant cells provided an assay for agents able to circumvent the tubulin alterations that result in acquired paclitaxel resistance.

BRCA1 regulates microtubule dynamics and taxane-induced apoptotic cell signaling.

The taxanes are effective microtubule-stabilizing chemotherapy drugs used in the treatment of various solid tumors. However, the emergence of drug resistance hampers their clinical efficacy. The molecular basis of clinical taxane resistance remains poorly understood. Breast cancer 1, early onset gene, BRCA1, is a tumor-suppressor gene, whose expression has been correlated with taxane sensitivity in many solid tumors including non-small cell lung cancer. However, the molecular mechanism underlying the relationship between BRCA1 (B1) expression and taxane activity remains unclear. To this end, we created a stable B1 knockdown A549 cell line (B1-KD) to investigate B1's role in microtubule biology and response to taxane treatment. We show that B1-KD rendered A549 cells resistant to paclitaxel (PTX), phenocopying clinical studies showing that low B1 expression correlated with taxane resistance. As previously reported, we show that loss of B1 enhanced centrosomal γ-tubulin localization and microtubule nucleation. Interestingly, we found that the B1-KD cells exhibited increased microtubule dynamics as compared with parental A549 cells, as assessed by live-cell confocal microscopy using enhanced green fluorescent protein-tagged α-tubulin or EB1 protein. In addition, we showed that loss of B1 impairs the ability of PTX to induce microtubule polymerization using immunofluorescence microscopy and a cell-based tubulin polymerization assay. Furthermore, B1-KD cells exhibited significantly lower intracellular binding of a fluorescently labeled PTX to microtubules. Recent studies have shown that PTX-stabilized microtubules serves as a scaffold for pro-caspase-8 binding and induction of apoptosis downstream of induced-proximity activation of caspase-8. Here we show that loss of B1 reduces the association of pro-caspase-8 with microtubules and subsequently leads to impaired PTX-induced activation of apoptosis. Taken together, our data show that B1 regulates indirectly endogenous microtubule dynamics and stability while its loss leads to microtubules that are more dynamic and less susceptible to PTX-induced stabilization conferring taxane resistance.

Activities of the microtubule-stabilizing agents epothilones A and B with purified tubulin and in cells resistant to paclitaxel (Taxol(R)).

Epothilones A and B, natural products with minimal structural analogy to taxoids, have effects similar to those of paclitaxel (Taxol(R)) in cultured cells and on microtubule protein, but differ from paclitaxel in retaining activity in multidrug-resistant cells. We examined interactions of the epothilones with purified tubulin and additional cell lines, including a paclitaxel-resistant ovarian carcinoma line with an altered beta-tubulin. The epothilones, like paclitaxel, induced tubulin to form microtubules at low temperatures and without GTP and/or microtubule-associated proteins. The epothilones are competitive inhibitors of the binding of [3H]paclitaxel to tubulin polymers. The apparent Ki values for epothilones A and B were 1.4 and 0.7 microM by Hanes analysis and 0.6 and 0.4 microM by Dixon analysis. In the paclitaxel-sensitive human cell lines we examined, epothilone B had greater antiproliferative activity than epothilone A or paclitaxel, while epothilone A was usually less active than paclitaxel. A multidrug-resistant colon carcinoma line and the paclitaxel-resistant ovarian line retained sensitivity to the epothilones. With Potorous tridactylis kidney epithelial (PtK2) cells examined by indirect immunofluorescence, microtubule bundles appeared more rapidly following epothilone B treatment, and there were different proportions of various mitotic aberrations following treatment with different drugs.

Effect of calcium channel blockers on the action of various antitumour agents in the yeast Saccharomyces cerevisiae.

The cytotoxic effects of a 22 h treatment with four antineoplastic agents in the yeast Saccharomyces cerevisiae ATCC 2366 were investigated. Two agents, doxorubicin and 5-fluorouracil (5-FU), were effective in decreasing the colony-forming ability of yeast cells. Following examination under the light microscope, the effect of doxorubicin appeared to be, at least partially, due to killing of yeast cells whereas the effect of 5-FU was rather due to changes in cell structure leading to abnormal bud formation. For amsacrine (AMSA) and melphalan, cytotoxicity was totally absent. In the presence of diltiazem the above described effects were not significantly changed. When verapamil was added in the culture medium the cytotoxic activity of doxorubicin and 5-FU did not change. However, following treatment with AMSA in combination with verapamil, cell survival was significantly decreased whereas the presence of verapamil increased the yeast survival which was observed after melphalan treatment.

Combinations of paclitaxel and vinblastine and their effects on tubulin polymerization and cellular cytotoxicity: characterization of a synergistic schedule.

Paclitaxel (PTX) and vinblastine (VBL) represent 2 classes of drugs that target tubulin but have separate binding properties and opposing mechanisms of action. To evaluate the potential use of these agents together in a chemotherapeutic regimen, we investigated their effects on the dynamics of tubulin polymerization and cellular cytotoxicity, when administered singly or in combination. In human epidermoid carcinoma KB cells and MCF-7 breast carcinoma cells, we observed a time- and dose-dependent effect on cytoskeletal dynamics for both PTX and VBL. Tubulin polymerization induced by PTX was stable for more than 24 hr. When PTX treatment was followed by VBL, a time- and dose-dependent reversal of tubulin polymerization was observed. In contrast, rapid tubulin polymerization occurred when VBL was followed by PTX. When both agents were added simultaneously, a diminution of PTX-induced tubulin polymerization was observed with increasing doses of VBL; a maximum reduction was achieved when equal concentrations were used. Examination of the tubulin pattern by immunofluorescence in MCF-7 breast cancer cells confirmed and extended our findings. Bundle formation followed treatment with PTX. Addition of increasing concentrations of VBL prevented bundling; however, the normal cytoskeletal architecture was not restored. Cytotoxicity studies carried out using the median dose effect principles and the combination index analysis showed synergism when VBL and PTX were administered sequentially and antagonism for simultaneous administration. Our results demonstrate changes in tubulin dynamics following drug treatment and provide a rationale for combined PTX/VBL therapy after careful evaluation of the schedule of administration.