A ubiquitous beta-tubulin disrupts microtubule assembly and inhibits cell proliferation

Vertebrate tubulin is encoded by a multigene family that produces distinct gene products, or isotypes, of both the alpha- and beta-tubulin subunits. The isotype sequences are conserved across species supporting the hypothesis that different isotypes subserve different functions. To date, however, most studies have demonstrated that tubulin isotypes are freely interchangeable and coassemble into all classes of microtubules. We now report that, in contrast to other isotypes, overexpression of a mouse class V beta-tubulin cDNA in mammalian cells produces a strong, dose-dependent disruption of microtubule organization, increased microtubule fragmentation, and a concomitant reduction in cellular microtubule polymer levels. These changes also disrupt mitotic spindle assembly and block cell proliferation. Consistent with diminished microtubule assembly, there is an increased tolerance for the microtubule stabilizing drug, paclitaxel, which is able to reverse many of the effects of class V beta-tubulin overexpression. Moreover, transfected cells selected in paclitaxel exhibit increased expression of class V beta-tubulin, indicating that this isotype is responsible for the drug resistance. The results show that class V beta-tubulin is functionally distinct from other tubulin isotypes and imparts unique properties on the microtubules into which it incorporates.     

Comparison of beta-tubulin mRNA and protein levels in 12 human cancer cell lines

Antimitotic drugs are chemotherapeutic agents that bind tubulin and microtubules. Resistance to these drugs is a major clinical problem. One hypothesis is that the cellular composition of tubulin isotypes may predict the sensitivity of a tumor to antimitotics. Reliable and sensitive methods for measuring tubulin isotype levels in cells and tissues are needed to address this hypothesis. Quantitative measurements of tubulin isotypes have frequently relied upon inferring protein amounts from mRNA levels. To determine whether this approach is justified, protein and mRNA levels of beta-tubulin isotypes from 12 human cancer cell lines were measured. This work focused on only beta-tubulin isotypes because we had readily available monoclonal antibodies for quantitative immunoblots. The percentage of beta-tubulin isotype classes I, II, III, and IVa + IVb mRNA and protein were compared. For beta-tubulin class I that comprises >50% of the beta-tubulin protein in 10 of the 12 cell lines, there was good agreement between mRNA and protein percentages. Agreement between mRNA and protein was also found for beta-tubulin class III. For beta-tubulin classes IVa + IVb, we observed higher protein levels compared to mRNA levels.Beta-tubulin class II protein was found in only four cell lines and in very low abundance. We conclude that quantitative Western blotting is a reliable method for measuring tubulin isotype levels in human cancer cell lines. Inferring protein amounts from mRNA levels should be done with caution, since the correspondence is not one-to-one for all tubulin isotypes.     

Intercellular transfer of the oncogenic receptor EGFRvIII by microvesicles derived from tumour cells

Aggressive human brain tumours (gliomas) often express a truncated and oncogenic form of the epidermal growth factor receptor, known as EGFRvIII. Within each tumour only a small percentage of glioma cells may actually express EGFRvIII; however, most of the cells exhibit a transformed phenotype. Here we show that EGFRvIII can be 'shared' between glioma cells by intercellular transfer of membrane-derived microvesicles ('oncosomes'). EGFRvIII expression in indolent glioma cells stimulates formation of lipid-raft related microvesicles containing EGFRvIII. Microvesicles containing this receptor are then released to cellular surroundings and blood of tumour-bearing mice, and can merge with the plasma membranes of cancer cells lacking EGFRvIII. This event leads to the transfer of oncogenic activity, including activation of transforming signalling pathways (MAPK and Akt), changes in expression of EGFRvIII-regulated genes (VEGF, Bcl-x(L), p27), morphological transformation and increase in anchorage-independent growth capacity. Thus, membrane microvesicles of cancer cells can contribute to a horizontal propagation of oncogenes and their associated transforming phenotype among subsets of cancer cells.     

Paclitaxel resistance in cells with reduced beta-tubulin

We previously described the isolation of colcemid resistant Chinese hamster ovary cell lines containing alpha- and beta-tubulin mutations that increase microtubule assembly and stability. By analyzing colcemid sensitive revertants from one of the beta-tubulin mutants, we now find that loss or inactivation of the mutant allele represents the most common mechanism of reversion. Consistent with this loss, the revertants have 35% less tubulin at steady state, no evidence for the presence of a mutant polypeptide, and a normal extent of tubulin polymerization. In addition to the loss of colcemid resistance, the revertant cells exhibit increased resistance to paclitaxel relative to wild-type cells. This paclitaxel resistance can be suppressed by transfecting the revertant cells with a cDNA for wild-type beta-tubulin, indicating that the reduction in tubulin in the revertant cells is responsible for the resistance phenotype. We propose that reducing tubulin levels may represent a novel mechanism of paclitaxel resistance.     

Mutations at leucine 215 of beta-tubulin affect paclitaxel sensitivity by two distinct mechanisms

Paclitaxel resistance mutations in Chinese hamster ovary cells frequently alter a cluster of leucine residues in the H6-H7 loop region of beta-tubulin. To gain further insight into the role of this region in microtubule assembly and drug resistance, site-directed mutagenesis was used to systematically change amino acid L215. The mutated genes were cloned into a tetracycline-regulated expression vector and transfected into wild-type cells. Most of the mutations destabilized microtubule assembly, causing a decreased fraction of tubulin to appear in the microtubule cytoskeleton. In each case, the decreased level of assembly was associated with paclitaxel resistance and increased colcemid sensitivity. In two cases, however, the alteration did not significantly perturb the level of assembled tubulin or confer resistance to paclitaxel. One of these, L215V, produced little or no detectable phenotype, while the other, L215I, conferred increased sensitivity to paclitaxel. The increased drug sensitivity did not extend to epothilone A, a drug that binds to the same site and has a mechanism of action similar to that of paclitaxel, or colcemid, a drug with an opposing mechanism of action and a distinct binding site. Moreover, L215I conferred enhanced paclitaxel sensitivity at very low levels of expression, and sensitivity was not further enhanced in cells with higher levels of expression, implying that paclitaxel acts substoichiometrically. These properties, along with the proximity of L215 to the drug binding site, suggests that the L215I substitution may enhance the binding or effectiveness of paclitaxel. Our studies confirm the importance of the H6-H7 loop of beta-tubulin in microtubule assembly and resistance to antimitotic drugs. They also identify the first mammalian mutation shown to specifically increase sensitivity to paclitaxel.     

Delineating the interaction of combretastatin A-4 with αβ tubulin isotypes present in drug resistant human lung carcinoma using a molecular modeling approach

Abstract

Tubulin isotypes are known to regulate microtubule dynamic instability and contribute to the development of drug resistance in certain types of cancers. Combretastatin-A4 (CA-4) has a potent anti-mitotic, vascular disrupting and anti-angiogenic activity. It binds at the interface of αβ tubulin heterodimers and inhibits microtubules assembly. Interestingly, the CA-4 resistant human lung carcinoma shows alteration of βI and βIII isotype levels, a higher expression of βI tubulin isotype and a decreased expression of βIII tubulin isotypes has been reported in drug resistant cell lines. However, the origin of CA-4 resistance in lung carcinoma is not well understood. Here, we investigate the interaction and binding affinities of αβI, αβIIb, αβIII and αβIVa tubulin isotypes with CA-4, employing molecular modeling approaches. Sequence analysis shows that variations in residue composition at the CA-4 binding pocket of βI, βIII and βIVa tubulin isotypes when compared to template βIIb isotype. Molecular docking result shows that the CA-4 prefers ‘cis’ conformation in all αβ-tubulin isotypes. Molecular dynamics simulation reveal role of H7 helix, T7 loop and H8 helix of β-tubulin in lower binding affinity of αβI and αβIII isotypes for CA-4. The order of binding energy for CA-4 is αβIIb?>?αβIVa?>?αβI?>?αβIII. This suggest that drug resistance is induced in human lung carcinoma cells by altering the expression of β-tubulin isotypes namely βI and βIII which show lowest binding affinities. Our present study can help in designing potential CA-4 analogs against drug-resistant cancer cells showing altered expression of tubulin isotypes. Abbreviations: CA-4 combretastatin-A4

MD molecular dynamics

RMSD root mean square deviation

DSSP dictionary of secondary structure of proteins

VMD visual molecular dynamics

Communicated by Ramaswamy H. Sarma     

Free intermingling of mammalian beta-tubulin isotypes among functionally distinct microtubules

Mammalian cells express a spectrum of tubulin isotypes whose relationship to the diversity of microtubule function is unknown. To examine whether different isotypes are segregated into functionally distinct microtubules, we generated immune sera capable of discriminating among the various naturally occurring beta-tubulin isotypes. Cloned fusion proteins encoding each isotype were used first to tolerogenize animals against shared epitopes, and then as immunogens to elicit a specific response. In experiments using these sera, we show that there is neither complete nor partial segregation of beta-tubulin isotypes: both interphase cytoskeletal and mitotic spindle microtubules are mixed copolymers of all expressed beta-tubulin isotypes. Indeed, a highly divergent isotype normally expressed only in certain hematopoietic cells is also indiscriminately assembled into all microtubules both in their normal context and when transfected into HeLa cells.     

BetaIII-tubulin induces paclitaxel resistance in association with reduced effects on microtubule dynamic instability

The development of resistance to paclitaxel in tumors is one of the most significant obstacles to successful therapy. Overexpression of the betaIII-tubulin isotype has been associated with paclitaxel resistance in a number of cancer cell lines and in tumors, but the mechanism of resistance has remained unclear. Paclitaxel inhibits cancer cell proliferation by binding to the beta-subunit of tubulin in microtubules and suppressing microtubule dynamic instability, leading to mitotic arrest and cell death. We hypothesized that betaIII-tubulin overexpression induces resistance to paclitaxel either by constitutively enhancing microtubule dynamic instability in resistant cells or by rendering the microtubules less sensitive to the suppression of dynamics by paclitaxel. Using Chinese hamster ovary cells that inducibly overexpress either betaI- or betaIII-tubulin, we analyzed microtubule dynamic instability during interphase by microinjection of rhodamine-labeled tubulin and time-lapse fluorescence microscopy. In the absence of paclitaxel, there were no differences in any aspect of dynamic instability between the two beta-tubulin-overexpressing cell types. However, in the presence of 150 nm paclitaxel, dynamic instability was suppressed to a significantly lesser extent (suppressed only 12%) in cells overexpressing betaIII-tubulin than in cells overexpressing similar levels of betaI-tubulin (suppressed 47%). The results suggest that overexpression of betaIII-tubulin induces paclitaxel resistance by reducing the ability of paclitaxel to suppress microtubule dynamics. The results also suggest that endogenous regulators of microtubule dynamics may differentially interact with individual tubulin isotypes, supporting the idea that differential expression of tubulin isotypes has functional consequences in cells.     

Effects of beta-2 microglobulin anti-sense oligonucleotides on sensitivity of HER2/neu oncogene-expressing and nonexpressing target cells to lymphocyte-mediated lysis.

The mechanism by which HER2/neu overexpressing tumor cells resist NK, LAK, and LDCC cytotoxic lymphocytes was investigated. Resistance was not explained by a delay in kinetics of lysis, concurrent resistance to TNF, or a diminished expression of the transferrin receptor. HLA-class I expression, however, was markedly elevated compared to HER2 nonexpressing targets suggesting a reason for resistance. To test the role of class I, we selectively decreased expression by incubation of targets with beta-2 microglobulin anti-sense oligonucleotides. Anti-sense-treated HER2+ targets, displaying levels of class I comparable to HER2- targets, were still markedly resistant to cytotoxic effectors. Down-regulation of class I expression in HER2- carcinoma cells also had no effect on sensitivity to cytotoxicity by anti-sense treatment of Raji and U937 targets resulted in enhanced sensitivity to NK and LAK effectors but not to T cells mediating LDCC. These data indicate resistance to cytotoxicity in HER2-expressing targets cannot be solely explained by heightened expression of class I. The data also support the concept that class I expression regulates sensitivity to NK and LAK cells (but not LDCC effectors) in selected targets.     

Rationalization of paclitaxel insensitivity of yeast ß-tubulin and human ßIII-tubulin isotype using principal component analysis

ABSTRACT: BACKGROUND: The chemotherapeutic agent paclitaxel arrests cell division by binding to the hetero-dimeric protein tubulin. Subtle differences in tubulin sequences, across eukaryotes and among beta-tubulin isotypes, can have profound impact on paclitaxel-tubulin binding. To capture the experimentally observed paclitaxel-resistance of human betaIII tubulin isotype and yeast beta-tubulin, within a common theoretical framework, we have performed structural principal component analyses of beta-tubulin sequences across eukaryotes. RESULTS: The paclitaxel-resistance of human betaIII tubulin isotype and yeast beta-tubulin uniquely mapped on to the lowest two principal components, defining the paclitaxel-binding site residues of beta-tubulin. The molecular mechanisms behind paclitaxel-resistance, mediated through key residues, were identified from structural consequences of characteristic mutations that confer paclitaxel-resistance. Specifically, Ala277 in betaIII isotype was shown to be crucial for paclitaxel-resistance. CONCLUSIONS: The present analysis captures the origin of two apparently unrelated events, paclitaxel-insensitivity of yeast tubulin and human betaIII tubulin isotype, through two common collective sequence vectors.     

Delivery of chemo-sensitizing siRNAs to HER2+-breast cancer cells using RNA aptamers

Human epidermal growth factor receptor 2 (HER2) expression in breast cancer is associated with an aggressive phenotype and poor prognosis, making it an appealing therapeutic target. Trastuzumab, an HER2 antibody-based inhibitor, is currently the leading targeted treatment for HER2(+)-breast cancers. Unfortunately, many patients inevitably develop resistance to the therapy, highlighting the need for alternative targeted therapeutic options. In this study, we used a novel, cell-based selection approach for isolating 'cell-type specific', 'cell-internalizing RNA ligands (aptamers)' capable of delivering therapeutic small interfering RNAs (siRNAs) to HER2-expressing breast cancer cells. RNA aptamers with the greatest specificity and internalization potential were covalently linked to siRNAs targeting the anti-apoptotic gene, Bcl-2. We demonstrate that, when applied to cells, the HER2 aptamer-Bcl-2 siRNA conjugates selectively internalize into HER2(+)-cells and silence Bcl-2 gene expression. Importantly, Bcl-2 silencing sensitizes these cells to chemotherapy (cisplatin) suggesting a potential new therapeutic approach for treating breast cancers with HER2(+)-status. In summary, we describe a novel cell-based selection methodology that enables the identification of cell-internalizing RNA aptamers for targeting therapeutic siRNAs to HER2-expressing breast cancer cells. The future refinement of this technology may promote the widespread use of RNA-based reagents for targeted therapeutic applications.     

Sequence of chicken c beta 7 tubulin. Analysis of a complete set of vertebrate beta-tubulin isotypes

In chicken, beta-tubulin is encoded by a family of seven genes. We have now isolated and sequenced overlapping cDNA clones corresponding to gene c beta 7 (previously designated c beta 4'), the only chicken beta-tubulin not previously characterized. The inferred amino acid sequence of c beta 7 tubulin is identical with the class I beta-tubulin isotype found in human, mouse and rat. Moreover, c beta 7 is highly expressed in almost all tissue and cell types in chicken, a pattern similar to those of the genes for class I beta-tubulin isotypes in other vertebrates. Comparison of the complete family of chicken beta-tubulin gene sequences reveals that the heterogeneity of beta-tubulin polypeptides encoded in a higher eukaryote is confined to six distinct beta-tubulin isotypes. Five of these are members of evolutionarily conserved isotypic classes (I to V), whereas the sixth represents a divergent erythroid-specific tubulin whose sequence has not been conserved.     

Axonal transport of class II and III beta-tubulin: evidence that the slow component wave represents the movement of only a small fraction of the tubulin in mature motor axons.

Pulse-labeling studies demonstrate that tubulin synthesized in the neuron cell body (soma) moves somatofugally within the axon (at a rate of several millimeters per day) as a well-defined wave corresponding to the slow component of axonal transport. A major goal of the present study was to determine what proportion of the tubulin in mature motor axons is transported in this wave. Lumbar motor neurons in 9-wk-old rats were labeled by injecting [35S]methionine into the spinal cord 2 wk after motor axons were injured (axotomized) by crushing the sciatic nerve. Immunoprecipitation with mAbs which recognize either class II or III beta-tubulin were used to analyze the distributions of radioactivity in these isotypes in intact and axotomized motor fibers 5 d after labeling. We found that both isotypes were associated with the slow component wave, and that the leading edge of this wave was enriched in the class III isotype. Axotomy resulted in significant increases in the labeling and transport rates of both isotypes. Immunohistochemical examination of peripheral nerve fibers demonstrated that nearly all of the class II and III beta-tubulin in nerve fibers is located within axons. Although the amounts of radioactivity per millimeter of nerve in class II and III beta-tubulin were significantly greater in axotomized than in control nerves (with increases of +160% and +58%, respectively), immunoassay revealed no differences in the amounts of these isotypes in axotomized and control motor fibers. We consider several explanations for this paradox; these include the possibility that the total tubulin content is relatively insensitive to changes in the amount of tubulin transported in the slow component wave because this wave represents the movement of only a small fraction of the tubulin in these motor fibers.     

Expression of cold-adapted beta-tubulins confer cold-tolerance to human cellular microtubules

Isolated microtubule proteins from the cold-adapted fish, Atlantic cod (Gadus morhua), assemble at temperatures between 8 and 30 degrees C, while avian and mammalian microtubules normally do not assemble at temperatures below 20 degrees C. Tubulin, the main component in microtubules, is expressed as many isotypes. Microtubules with different isotype composition have been shown to have different dynamic properties in vitro. Our hypothesis was that cold-tolerance of microtubules is caused by tubulin isotypes that differ in the primary sequence compared to mammalian tubulins. Here we show that transfection of human HepG2 cells with cod beta-tubulin induced cold-adaptation of the endogenous microtubules. Incorporation of one single tubulin isotype can induce cold-tolerance to cold-intolerant microtubules. Three cod beta-tubulin isotypes were tested and two of these (beta1 and beta2) transferred cold-tolerance to HepG2 microtubules, thus not all cod beta-tubulins were able to confer cold-stability.     

Intra-allelic suppression of a mutation that stabilizes microtubules and confers resistance to colcemid

Cmd 4 is a colcemid resistant beta-tubulin mutant of Chinese hamster ovary cells that exhibits hypersensitivity to paclitaxel and temperature sensitivity for growth. The mutant beta-tubulin allele in this cell line encodes a D45Y amino acid substitution that produces colcemid resistance by making microtubules more stable. By selecting revertants of the temperature sensitive and paclitaxel hypersensitive phenotypes, we have identified three cis-acting suppressors of D45Y. One suppressor, V60A, maps to the same region as the D45Y alteration, and a second suppressor, Q292H, maps to a distant location. Both appear to produce compensatory changes in microtubule assembly that counteract the effects of the original D45Y substitution. Consistent with this view, expression of the V60A mutation in transfected wild-type cells produced paclitaxel resistance and greatly decreased microtubule assembly. Additionally, it produced a paclitaxel-dependent phenotype in which cells grew normally in the presence, but not the absence, of the drug. The Q292H mutation caused even greater disassembly of microtubules such that cells were unable to proliferate when the transgene was expressed; but, unlike the V60A mutation, cell growth could not be rescued by paclitaxel. A third suppressor, A254V, maps to a region near the interface between alpha- and beta-tubulin that contains the colchicine binding site. Although it made transfected wild-type cells hypersensitive to colcemid, it did not affect paclitaxel or vinblastine sensitivity, nor did it reduce microtubule assembly. We suggest that this mutation acts by increasing tubulin's affinity for colcemid.     

Differential sorting of beta tubulin isotypes into colchicine-stable microtubules during neuronal and muscle differentiation of embryonal carcinoma cells.

Pluripotent P19 embryonal carcinoma (EC) cells were differentiated along the neuronal and muscle pathways. Comparisons of class I, II, III, and IV beta tubulin isotypes in total and colchicine-stable microtubule (MT) arrays from uncommitted EC, neuronal, and muscle cells were made by immunoblotting and by indirect immunofluorescence microscopy. In undifferentiated EC cells the relative amounts of these four isotypes are the same in both the total and stable MT populations. Subcellular sorting of beta tubulin isotypes was demonstrated in both neuronal and muscle differentiated cells. During neuronal differentiation, class II beta tubulin is preferentially incorporated into the colchicine-stable MTs while class III beta tubulin is preferentially found in the colchicine-labile MTs. The subcellular sorting of class II into stable MTs correlates with the increased staining of MAP 1B, and with the expression of MAP 2C and tau. Although muscle differentiated cells express class II beta tubulin, stable MTs in these cells do not preferentially incorporate this isotype but instead show increased incorporation of class IV beta tubulin. Muscle cells do not show high levels of MAP 1B and do not express MAP 2C or tau. These results are consistent with the hypothesis that a subcellular sorting of tubulin isotypes is the result of a complex interaction between tubulin isotypes and MT-associated proteins.