Β-tubulin Isotypes Research Articles

Rationalization of paclitaxel insensitivity of yeast β-tubulin and human βIII-tubulin isotype using principal component analysis

BackgroundThe chemotherapeutic agent paclitaxel arrests cell division by binding to the hetero-dimeric protein tubulin. Subtle differences in tubulin sequences, across eukaryotes and among β-tubulin isotypes, can have profound impact on paclitaxel-tubulin binding. To capture the experimentally observed paclitaxel-resistance of human βIII tubulin isotype and yeast β-tubulin, within a common theoretical framework, we have performed structural principal component analyses of β-tubulin sequences across eukaryotes.ResultsThe paclitaxel-resistance of human βIII tubulin isotype and yeast β-tubulin uniquely mapped on to the lowest two principal components, defining the paclitaxel-binding site residues of β-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 βIII isotype was shown to be crucial for paclitaxel-resistance.ConclusionsThe present analysis captures the origin of two apparently unrelated events, paclitaxel-insensitivity of yeast tubulin and human βIII tubulin isotype, through two common collective sequence vectors.

Long-term exposure to imatinib reduced cancer stem cell ability through induction of cell differentiation via activation of MAPK signaling in glioblastoma cells

Glioblastoma multiforme (GBM) was shown to harbor therapy-resistant cancer stem cells that were major causes of recurrence. PDGFR (platelet-derived growth factor receptor) and c-Kit (stem cell factor receptor) signaling play important roles in initiation and maintenance of malignant glioma. This study demonstrated that long-term culture with imatinib mesylate, the tyrosine kinase inhibitor against PDGFR and c-Kit resulted in reduced cancer stem cell ability in glioblastoma cells through cell differentiation. Derived from RG glioblastoma cells co-cultured with imatinib for 3 months, RG-IM cells showed distinct properties of cell cycle distribution and morphology in addition to significantly decreased ability to form aggregates and colonies in vitro and tumorigenicity in vivo. Increased expression of GFAP (astrocyte marker) and class III β-tubulin isotype (Tuj1, neuron marker) were detected with morphology like neurons or astrocytes in RG-IM cells. Furthermore, decreased expression of stem cell markers, i.e., CD133, Oct-3/4, nestin, and Bmi1, and increased terminal neural cell markers, GFAP, Tuj1, etc., were identified in RG-IM at the mRNA level. All these markers were changed in RG cells when PDGFRB and c-Kit expression were double knocked down by siRNA. Cell differentiation agent, all-trans retinoic acid (ATRA) caused similar effect as that with imatinib in RG cells, while adding PDGF-B and SCF in RG-IM resulted in cell dedifferentiation to some extent. Moreover, differentiation in RG cells treated by imatinib or ATRA was mainly driven by MAPK signaling pathways. In summary, continuous inhibition on PDGFR and c-Kit signaling disturbed glioma stem cells biology in subsets of GBM cells and may have potentials in clinical applications.

Characterization of a human βV‐tubulin antibody and expression of this isotype in normal and malignant human tissue

There are seven distinct β-tubulin isotypes and eight α-tubulin isotypes in mammals that are hypothesized to have tissue- and cell-specific functions. There is an interest in the use of tubulin isotypes as prognostic markers of malignancy. βV-tubulin, like βIII-tubulin, has been implicated in malignant transformation and drug resistance, however little is known about its localization and function. Thus, we generated for the first time, a rabbit polyclonal antibody specific for human βV-tubulin. The antibody did not cross-react with mouse βV-tubulin or other human β-tubulin isotypes and specifically labeled βV-tubulin by immunoblotting, immunofluorescence and immunohistochemistry. Immunohistochemistry of various human normal tissues revealed that βV-tubulin was expressed in endothelial cells, myocytes and cells with muscle differentiation, structures with transport and/or secretory function such as renal tubules, pancreatic ducts and bile ducts, and epithelium with secretory function such as prostate. βV-tubulin was also specifically expressed in pancreatic islets and intratubular germ cell neoplasia, where it may have diagnostic utility. Initial studies in breast, lung and ovarian cancers indicated aberrant expression of βV-tubulin, suggesting that this isoform may be associated with tumorigenesis. Thus, βV-tubulin expression is a potentially promising prognostic marker of malignancy.

Peloruside, Laulimalide, and Noscapine Interactions with Beta-Tubulin

This article reviews the recent findings regarding the binding sites, binding modes and binding affinities of three novel antimitotic drugs peloruside, laulimalide and noscapine with respect to tubulin as the target of their action. These natural compounds are shown to bind to β-tubulin and stabilize microtubules for the cases of peloruside A and laulimalide, and prolong the time spent in pause for noscapine. Particular attention is focused on β-tubulin isotypes as targets for new cancer chemotherapy agents and the amino acid differences in the binding site for these compounds between isotypes. We propose a new strategy for antimitotic drug design that exploits differential distributions of tubulin isotypes between normal and cancer cells and corresponding differential affinities between various drug molecules and tubulin isotypes.

Distinct Functional Roles of β-Tubulin Isotypes in Microtubule Arrays of Tetrahymena thermophila, a Model Single-Celled Organism

BackgroundThe multi-tubulin hypothesis proposes that each tubulin isotype performs a unique role, or subset of roles, in the universe of microtubule function(s). To test this hypothesis, we are investigating the functions of the recently discovered, noncanonical β-like tubulins (BLTs) of the ciliate, Tetrahymena thermophila. Tetrahymena forms 17 distinct microtubular structures whose assembly had been thought to be based on single α- and β-isotypes. However, completion of the macronuclear genome sequence of Tetrahymena demonstrated that this ciliate possessed a β-tubulin multigene family: two synonymous genes (BTU1 and BTU2) encode the canonical β-tubulin, BTU2, and six genes (BLT1-6) yield five divergent β-tubulin isotypes. In this report, we examine the structural features and functions of two of the BLTs (BLT1 and BLT4) and compare them to those of BTU2.Methodology/Principal FindingsWith respect to BTU2, BLT1 and BLT4 had multiple sequence substitutions in their GTP-binding sites, in their interaction surfaces, and in their microtubule-targeting motifs, which together suggest that they have specialized functions. To assess the roles of these tubulins in vivo, we transformed Tetrahymena with expression vectors that direct the synthesis of GFP-tagged versions of the isotypes. We show that GFP-BLT1 and GFP-BLT4 were not detectable in somatic cilia and basal bodies, whereas GFP-BTU2 strongly labeled these structures. During cell division, GFP-BLT1 and GFP-BLT4, but not GFP-BTU2, were incorporated into the microtubule arrays of the macronucleus and into the mitotic apparatus of the micronucleus. GFP-BLT1 also participated in formation of the microtubules of the meiotic apparatus of the micronucleus during conjugation. Partitioning of the isotypes between nuclear and ciliary microtubules was confirmed biochemically.Conclusion/SignificanceWe conclude that Tetrahymena uses a family of distinct β-tubulin isotypes to construct subsets of functionally different microtubules, a result that provides strong support for the multi-tubulin hypothesis.

A novel mutation in the β‐tubulin gene TUBB2B associated with complex malformation of cortical development and deficits in axonal guidance

Neurological disorders characterized by abnormal neuronal migration, organization, axon guidance, and maintenance have recently been associated with missense and splice-site mutations in the genes encoding α- and β-tubulin isotypes TUBA1A, TUBB2B, TUBB3, and TUBA8. We found a novel heterozygous mutation c.419G > C in exon 4 of the gene encoding TUBB2B in a female with microcephaly, agenesis of the corpus callosum, open-lip schizencephaly of the left parietal lobe, extensive polymicrogyria, basal ganglia and thalami dysmorphisms, and vermis and right third nerve hypoplasia. The missense change results in a glycine to alanine substitution; the mutated residue falls within an invariant glycine-rich region and therefore is likely to result in impaired protein function and possibly microtubule formation. This study expands the spectrum of brain malformations associated with mutations in the β-tubulin gene TUBB2B, supporting its critical role in migration/organization and axon guidance processes. In addition, it suggests a possible genetic aetiology of schizencephaly, thus strengthening the hypothesis that there is a common pathophysiological base in polymicrogyria and schizencephaly.

Tubulins as Therapeutic Targets in Cancer: from Bench to Bedside

Tubulin is the target of some of the most widely used and time-honored anticancer tubulin-binding agents (TBAs). The clinical usefulness of many TBAs has been held back as a result of tumor cell drug-resistance. The elucidation of the three-dimensional structure of αβ-tubulin dimer has provided an opportunity for rational drug design aimed at generating compounds that will target tubulin in therapeutically more efficacious ways compared to presently available drugs. An issue to be addressed is which one(s) of the tubulin species, their isotypes, or their posttranslationally modified forms, should be specifically targeted in cancer chemotherapy. This review offers a critical appraisal of current knowledge on tubulins in cancer and an update on new anti-neoplastic microtubule-targeted treatment strategies. Specifically, it examines, across disciplines, cellular/molecular, biochemical, clinical/pathological, and pharmacological aspects of β-tubulin isotypes, posttranslational modifications of tubulin dimers, γ-tubulin and microtubule nucleation, and microtubule regulatory proteins. Emphasis is placed on the overexpression of (i) the βIII isotype, which functions as a survival factor associated with dynamic instability of microtubules; (ii) γ-tubulin, a key microtubule nucleating protein; and (iii) the microtubule severing enzyme spastin, involved in cell motility and proliferation of glioblastoma cells. The role of βIII-tubulin in resistance of cancer cells to taxanes is examined. Attention is called to the novel concept that βIII-tubulin functions as a "gateway" for prosurvival signals in partnership with GTPases, such as GBP1. Appraisal is also offered on epothilones and the concept of hypersensitization to TBAs as promising therapeutic strategies in taxane resistant epithelial cancers and in high-grade gliomas.

Genetic diversity patterns of Haemonchus placei and Haemonchus contortus populations isolated from domestic ruminants in Brazil

Parasitic nematodes of the genus Haemonchus infect a range of ruminant hosts and are of major veterinary and economic importance. In this study, the genetic variability of seven isolates of Haemonchus placei and Haemonchus contortus was evaluated using the mitochondrial gene cytochrome oxidase subunit I and the nuclear gene β-tubulin isotype 1. A total of 156 specimens were obtained from cattle, sheep, goat and buffalo herds raised on commercial properties from the southern and southeastern regions of Brazil and identified to the species level by sequencing of the nuclear internal transcribed spacer 2. Thirty-four percent of the specimens were identified as H. placei and 66% as H. contortus. Cattle were the preferred hosts for H. placei, whereas H. contortus was most frequent in the other three ruminant species. Analysis of genetic differentiation between isolates revealed that high rates of gene flow are operating among populations of both nematode species, including among those from different ruminant host species. Populations of H. placei were less polymorphic and presented a lower frequency of single nucleotide polymorphisms associated with benzimidazole resistance compared with H. contortus. In line with the low amount of genetic structure observed among isolates, alleles of β-tubulin1 associated with benzimidazole resistance were present at relatively high frequencies of 5–20% in isolates of H. contortus from farms that never used this class of anthelmintic. The results presented here are consistent with the hypothesis of multiple origins of alleles associated with benzimidazole resistance, with the trade of animals among properties acting as the main factor promoting the spread of anthelmintic resistance.

Structural thermal adaptation of β‐tubulins from the Antarctic psychrophilic protozoan Euplotes focardii

Tubulin dimers of psychrophilic eukaryotes can polymerize into microtubules at 4°C, a temperature at which microtubules from mesophiles disassemble. This unique capability requires changes in the primary structure and/or in post-translational modifications of the tubulin subunits. To contribute to the understanding of mechanisms responsible for microtubule cold stability, here we present a computational structural analysis based on molecular dynamics (MD) and experimental data of three β-tubulin isotypes, named EFBT2, EFBT3, and EFBT4, from the Antarctic protozoon Euplotes focardii that optimal temperature for growth and reproduction is 4°C. In comparison to the β-tubulin from E. crassus, a mesophilic Euplotes species, EFBT2, EFBT3, and EFBT4 possess unique amino acid substitutions that confer different flexible properties of the polypeptide, as well as an increased hydrophobicity of the regions involved in microtubule interdimeric contacts that may overcome the microtubule destabilizing effect of cold temperatures. The structural analysis based on MD indicated that all isotypes display different flexibility properties in the regions involved in the formation of longitudinal and lateral contacts during microtubule polymerization. We also investigated the role of E. focardii β-tubulin isotypes during the process of cilia formation. The unique characteristics of the primary and tertiary structures of psychrophilic β-tubulin isotypes seem responsible for the formation of microtubules with distinct dynamic and functional properties.

Expression of β‐tubulin isotypes in classical Hodgkin's lymphoma

Microtubules consist of heterodimers of α- and β-tubulin. Aberrant expression of specific β-tubulin isotype is associated with resistance to chemotherapy in malignant tumors. In this study, we examined the expressions of β-tubulin isotypes in classical Hodgkin's lymphoma (cHL) by immunohistochemistry. Among the β-tubulin isotypes, class II β-tubulin (31/34, 91%) was most frequently overexpressed in the cytoplasm of almost all Hodgkin's and Reed-Sternberg (HRS) cells, followed by class I β-tubulin (18/34, 53%) and class III β-tubulin (12/34, 35%). Class IV β-tubulin was not expressed in any cHL case. Class I β-tubulin was expressed in the background lymphoid cells as well as the HRS cells. Thus, our results indicate that class II β-tubulin may be very useful for immunohistochemical diagnosis of cHL, and provide valuable information for the potential application of β-tubulin isotype-specific targeting.

A proteomic analysis of differential cellular responses to the short-chain fatty acids butyrate, valerate and propionate in colon epithelial cancer cells

The short chain fatty acids (SCFAs) are inhibitors of histone deacetylases (HDACi); they are produced naturally in the colon by fermentation. They affect cellular processes at a molecular and transcriptional level, the mechanisms of which may involve large numbers of proteins and integrated pathways. Butyrate is the most biologically potent of the SCFAs in colon epithelial cells, inhibiting human colon carcinoma cell proliferation and inducing apoptosis in vitro. In order to investigate the hypothesis that propionate and valerate possess unique and independent actions from butyrate, we combined proteomic and cellomic approaches for large-scale comparative analysis. Proteomic evaluation was undertaken using an iTRAQ tandem mass-spectrometry workflow and high-throughput High-content Analysis microscopy (HCA) was applied to generate cellomic information on the cell cycle and the cytoskeletal structure. Our results show that these SCFAs possess specific effects. Butyrate was shown to have more pronounced effects on the keratins and intermediate filaments (IFs); while valerate altered the β-tubulin isotypes' expression and the microtubules (MTs); propionate was involved in both mechanisms, displaying intermediate effects. These data suggest distinct physiological roles for SCFAs in colon epithelial function, offering new possibilities for cancer therapeutics.

Class III β‐tubulin and the cytoskeletal gateway for drug resistance in ovarian cancer

The Class III β-tubulin isotype (βIII-tubulin) is a predictive biomarker in ovarian cancer and other solid tumor malignancies. We discovered that βIII-tubulin function is linked to two GTPases: guanylate-binding protein 1 (GBP1), which activates its function, and GNAI1, which inhibits it. This finding was demonstrated in a panel of ovarian cancer cells resistant to several chemotherapeutic agents. Using a protein microarray, we identified PIM1 as the downstream partner of GBP1, recruited into the cytoskeleton under hypoxic conditions. The clinical value of these observations was tested by performing an archive study of 98 ovarian cancer patients, which demonstrated that the βIII-tubulin -/PIM1- cohort responded to treatment, exhibiting long overall survival (OS), while βIII-tubulin +/PIM+ patients experienced poor outcomes and OS times similar to patients receiving palliation alone. βIII-tubulin expression is commonly believed responsible for paclitaxel resistance due to its enhancement of the dynamic instability of microtubules, which counteracts the activity of taxanes. In contrast, our research reveals that βIII-tubulin behaves as a gateway for prosurvival signals, such as PIM1, to move into the cytoskeleton. When cells are exposed to microenvironmental stressors, they activate this pathway by telling the cytoskeleton to incorporate PIM1 through GBP1 and βIII-tubulin, which ultimately leads to drug resistance. This discovery reveals that βIII-tubulin does not act alone but requires partners to play its role. The discovery of such protein:protein interactions underlying this prosurvival cascade makes feasible the development of therapeutic approaches using novel compounds that are capable of inhibiting the transmission of prosurvival signals into the cytoskeleton.

Relationship between increased albendazole systemic exposure and changes in single nucleotide polymorphisms on the β-tubulin isotype 1 encoding gene in Haemonchus contortus

Resistance to benzimidazole anthelmintics in the nematode Haemonchus contortus has been correlated with single nucleotide polymorphisms (SNPs) on the β-tubulin isotype 1 gene. Three mutations can be used as markers for the detection of resistance, namely SNPs at position 200 and 167 (both TTC to TAC) or at position 198 (GAA to GCA). Harbouring a resistance genotype at any one of these codons can lead to a resistant phenotype. Our objective in this study was to analyse the frequencies of the three mutations when the albendazole dose rate and selection pressure were increased. We used adult H. contortus (males and females) collected directly from the abomasum of untreated lambs, or lambs treated with the manufacturer's recommended dose rate (5mg/kg), three times the recommended dose rate (15mg/kg), or nine times the recommended dose rate (45mg/kg). Anthelmintic efficacy was determined by worm and egg count reductions. For the surviving worms of the four treatment groups, the frequencies of each resistance SNP at codons 167, 200 and 198 were measured using pyrosequencing. Our results showed a strong relationship between an increasing dose rate and an increase in the frequency of the (TAC)200 SNP and a decrease in the (TAC)167 SNP. All worms genotyped were GAA at codon 198.

Immune Thrombocytopenia (ITP) and Hβ-1 Tubulin: The Arg307 His Substitution Leads to Increased Treatment Failures and Increased Platelet Turnover

Abstract 525▪▪This icon denotes a clinically relevant abstractIntroduction. Hβ-1 tubulin is a β-tubulin isotype required for platelet production and discoid shape. It is the only tubulin isotype with non-synonymous single nucleotide polymorphisms (SNPs), some of which lead to changes in platelet number, shape, and function. We have previously reported that in ITP, the R307H substitution in Hβ-1 tubulin is associated with more severe thrombocytopenia and the use of significantly more treatment modality types during treatment of ITP. It was not known, however, if the effects of R307H in ITP occurs via alteration in platelet production and/or turnover, and whether treatment outcomes are affected by the SNP.Methods. We sequenced exon 4 of TUBB1 (encoding Hβ-1 tubulin) using genomic DNA extracted from whole blood of 200 ITP patients to determine the presence of rs6070697 which leads to the the substitution of arginine for histidine at amino acid 307 (R307H). The first 100 patients in this series were the basis of our previous reports. Genotypes are reported as wild type (R/R), heterozygote (R/H), and homozygote SNP (H/H). A retrospective analysis of demographics and disease characteristics was performed for all patients. Patient responses to treatment modalities—classified as TPO-mimetic (TPO-m; eltrombopag, romiplostim, AKR-501), and non-TPO-mimetic treatments (non-TPO-m; all others)—were grouped by platelet counts (No Response (NR): 0–30; Partial Response (PR):31-100; Complete Response (CR):101-450). For a subset of patients, the absolute immature platelet fraction (A-IPF; immature platelet fraction (%) × platelet count) was also determined. The A-IPF is directly correlated with platelet production and turnover. The mean A-IPF for patients within each Hβ-1 genotype group was calculated and subsequently correlated with patient response to type of treatment. Fisher’s exact test was used to compare platelets at initial presentation, number of treatment modalities, and response to treatments between genotypes; two-tailed Student’s t-test was used to compare A-IPF measurements between genotypes.Results. Baseline demographics and disease characteristics were not significantly different between the three Hβ-1 genotypes, including sex, race, age at ITP onset, median follow-up of ITP, or presence of other autoimmune diseases. We found that homozygote SNP patients (H/H) presented with more severe thrombocytopenia requiring immediate treatment (platelet <30 × 109/L: H/H 100%, R/H 43%, R/R 64%, p=0.01). Median number of treatment modality types required was significantly higher for H/H patients compared to R/H and R/R (7.5, 5, and 4 treatment types, respectively; p=0.001). R/H and H/H patients had significantly higher (doubled) rate of treatment failures with non-TPO-m treatments compared to R/R patients (Failure rates 31% for R/H and H/H, 18% for R/R; Hazard Ratio 0.57; 95%CI 0.4–0.8; p=0.004). Furthermore, median A-IPFs were significantly higher in the R/H and H/H genotypes compared to the R/R group when a CR was achieved using non-TPO-m treatments (A-IPF=11.4 × 109/L for R/H and H/H, 6.7 × 109/L for R/R, p=0.005). In addition, the mean peak A-IPFs were significantly higher in the R/H and H/H genotypes compared to the R/R (20 × 109/L for R/H and H/H, 10 × 109/L for R/R, p=0.006). In contrast, when TPO-m were used we did not observe any significant difference between patient responses or A-IPFs across genotypes.Conclusion. Hβ-1 tubulin R307H substitution was strongly associated with double the rate of treatment failures to non-TPO-m modalities. This contrasts with TPO-m treatments where no difference was found. Mechanistically, it appears that the R307H substitution leads to increased platelet turnover in ITP patients, as evidenced by a significantly higher immature platelet number at normal platelet counts in patients with the substitution when non-TPO-m modalities were used. TPO-m treatments appear to overcome the differences imparted by the substitution. The high prevalence of the R307H SNP in the general population (30% for R/H and H/H) together with the strong clinical association with treatment failure to non-TPO-m treatments in ITP treatment suggest that this Hβ-1 SNP may be a predictive biomarker to guide clinical decision making. Disclosures:Bussel:Portola: Consultancy; Eisai: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; GlaxoSmithKline: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Amgen: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Cangene: Research Funding; Genzyme: Research Funding; Immunomedics: Research Funding; Ligand: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Shionogi: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Sysmex: Research Funding.

Abstract C215: Depletion of the resistance-associated βIII tubulin isotype enhances eribulin's potency in suppressing microtubule dynamic instability and promotes inhibition of both microtubule growth and shortening.

Abstract Eribulin mesylate (Halaven®) is a synthetic analog of the microtubule-targeted natural product halichondrin B, originally isolated from the marine sponge, Halichondria okadai. Recently, the US FDA approved eribulin mesylate for the treatment of patients with metastatic breast cancer who have previously received at least two chemotherapeutic regimens for the treatment of metastatic disease. Prior therapy should have included an anthracycline and a taxane in either the adjuvant or metastatic setting. Seven β-tubulin isotypes have been identified, and they are differentially expressed in a variety of tissues and tumors. A high level of βIII-tubulin often correlates with poor clinical outcome in several human tumors and with resistance to microtubule-targeted agents in certain human cancer cell lines and tumors. Here, we report the strong preferential suppression by eribulin of microtubule dynamic instability of purified microtubules depleted of βIII-tubulin in vitro. Tubulin was purified from bovine brain by cycles of temperature-dependent assembly and disassembly followed by phosphocellulose chromatography, and depleted of III-tubulin by immunoaffinity chromatography. Depletion of βIII-tubulin did not affect the inherent dynamic instability of the microtubules. Eribulin (100 nM) suppressed dynamic instability of βIII-tubulin-depleted microtubules significantly more strongly than it suppressed dynamic instability of microtubules assembled from unfractionated tubulin. Specifically, with microtubules composed of unfractionated tubulin, eribulin significantly suppressed only the growth rate (by 27%) and dynamicity (by 36%). The shortening rate and the catastrophe and rescue frequencies were suppressed only insignificantly (by 14%, 17%, and −2%, respectively). However, with βIII-tubulin-depleted microtubules, eribulin significantly suppressed almost all dynamic instability parameters including the growth rate (40%), shortening rate (42%), and the catastrophe and rescue frequencies (by 49% and 33%, respectively) leading to an overall suppression of microtubule dynamicity of 58%. Determination of the binding of eribulin to βIII- tubulin-depleted microtubules and to unfractionated microtubules is in progress. Acknowledgment: Supported by a grant from Eisai Inc. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr C215.

TGF-β-stimulated aberrant expression of class III β-tubulin via the ERK signaling pathway in cultured retinal pigment epithelial cells

The class III β-tubulin isotype (βIII) is expressed exclusively by neurons within the normal human retina and is not present in normal retinal pigment epithelial (RPE) cells in situ or in the early phase of primary cultures. However, aberrant expression of class III β-tubulin has been observed in passaged RPE cells and RPE cells with dedifferentiated morphology in pathologic epiretinal membranes from idiopathic macular pucker, proliferative vitreoretinopathy (PVR) and proliferative diabetic retinopathy (PDR). Transforming growth factor-β (TGF-β) has been implicated in dedifferentiation of RPE cells and has a critical role in the development of proliferative vitreoretinal diseases. Here, we investigated the potential effects of TGF-β on the aberrant expression of class III β-tubulin and the intracellular signaling pathway mediating these changes. TGF-β-induced aberrant expression and O-linked-β-N-acetylglucosamine (O-GlcNac) modification of class III β-tubulin in cultured RPE cells as determined using Western blotting, RT-PCR and immunocytochemistry. TGF-β also stimulated phosphorylation of ERK. TGF-β-induced aberrant expression of class III β-tubulin was significantly reduced by pretreatment with U0126, an inhibitor of ERK phosphorylation. Our findings indicate that TGF-β stimulated aberrant expression of class III β-tubulin via activation of the ERK signaling pathway. These data demonstrate that mature RPE cells have the capacity to express a neuron-associated gene in response to TGF-β stimulation and provide useful information towards understanding the pathogenesis of proliferative vitreoretinal diseases.

Targeting &amp;#946;III-Tubulin in Glioblastoma Multiforme: From Cell Biology and Histopathology to Cancer Therapeutics

Glioblastoma multiforme (GBM) is the most common, aggressive, and chemorefractory brain tumor in human adults. Notwithstanding significant discoveries in the elucidation of pathways of molecular signaling and genetics of GBM during the past 20 years there has been no breakthrough in the pharmacological treatment of this high-grade malignancy. We, and others, have previously demonstrated increased expression of βIII-tubulin in GBM asserting a link between aberrant expression of this β-tubulin isotype and a disruption of microtubule dynamics associated either with malignant tumor development de novo, or with progression and malignant transformation of a low-grade glioma into GBM. This article reviews βIII-tubulin as a promising target in the experimental treatment of GBM and examines the potential use of epothilones, a new family of anticancer agents shown to be active in βIII-tubulin-expressing tumor cells, as well as the "double hit" therapeutic concept of tumor cell sensitization to tubulin binding agents (TBAs) by βIII-tubulin silencing. The latest progress regarding the function and potential role of βIII-tubulin in aggressive tumor behavior, cancer stem cells, tumor cell hypoxia, and resistance to taxane-related compounds, is also critically appraised.

Candidate anthelmintic resistance-associated gene expression and sequence polymorphisms in a triple-resistant field isolate of Haemonchus contortus

An isolate of Haemonchus contortus, UGA/2004, highly resistant to benzimidazoles, levamisole, and ivermectin was isolated from sheep at the University of Georgia, and passaged through experimentally infected goats. We measured the expression of twenty-nine mRNAs encoding drug targets and P-glycoproteins (P-gps), comparing the results to a fully susceptible laboratory passaged isolate. Expression levels of some nicotinic acetylcholine receptor mRNAs were markedly different in UGA/2004. Levels of the Hco-acr-8b mRNA, encoding a truncated subunit, were very high in resistant L3, but undetectable in susceptible larvae, with expression of the full-length Hco-acr-8a mRNA also significant increased. Expression of Hco-unc-63 and Hco-unc-29.3 mRNAs was significantly reduced in the resistant larvae. Expression of the Hco-glc-3 and Hco-glc-5 mRNAs, encoding glutamate-gated chloride channel subunits, were slightly reduced in resistant larvae. We observed significant increases in the expression of the Hco-pgp-2 and Hco-pgp-9 mRNAs in the UGA/2004 larvae, consistent with previous reports; we also saw a decrease in the levels of Hco-pgp-1 mRNA. Treatment of the larvae with ivermectin and moxidectin in vitro produced variable and inconsistent changes in P-gp mRNA levels. The sequences of the β-tubulin isotype 1 mRNAs showed that the resistant larvae had a resistance-associated allele frequency of >95% at codon 200 and ∼40% and codon 167. No changes at codon 198 were present. The presence of the truncated acr-8b mRNA may be a reliable indicator of levamisole resistance, but complex changes in gene expression associated with macrocyclic lactone resistance make the identification of a single genetic marker for this resistance difficult.

The distribution of β-tubulin isotypes in cultured neurons from embryonic, newborn, and adult mouse brains

Tubulin, the subunit protein of microtubules, is an α/β heterodimer. Both α- and β-tubulin exist as numerous isotypes, differing in their amino acid sequences and encoded by different genes. The differences are highly conserved in evolution, suggesting that they are functionally significant. Neurons are a potentially very useful system for elucidating this significance, because they are highly differentiated cells and rich in tubulin isotypes. We have examined the distribution of β-tubulin isotypes in mouse primary cultured cortical neurons from embryonic fetus, newborn pups and adults. Neurons from both embryonic and adult mouse brains express the βI, βII, and βIII isotypes, but apparently not βIV or βV. βI, βII, and βIII are found in both cell bodies and neurites. However, the situation is different in newborn mice. Although βI and βIII are present in these neurons in both cell bodies and neurites and βIV is absent, just like in embryonic and adult mice, two striking differences were noted in the neurons from newborn mice. First, βV is apparently present evanescently in the neurons in both cell bodies and neurites. Interestingly, the βV was expressed strongly in newborn neurons after one day of culture; expression became much weaker after 3 days, and almost disappeared after 5 days. Second, the distribution of βII is different from other isotypes. After newborn mouse neurons were cultured for 3 days, βII started to disappear partly from the cell bodies; this was much more pronounced after five days in culture. Our findings suggest that βII's major function may involve the neurites and not the cell body. They also raise the possibility that βV has a unique role in the neurons of newborn mice.

P.11 The phenotype in E410K Beta-tubulin isotype 3 mutations

IntroductionThe Congenital Cranial Dysinnervation Disorders are congenital, non-progressive disorders that result from developmental abnormalities of one or more cranial nerves/nuclei with primary or secondary dysinnervation and include Duane's syndrome, Möbius...