Β-tubulin Isotypes Research Articles

Peloruside- and Laulimalide-Resistant Human Ovarian Carcinoma Cells Have βI-Tubulin Mutations and Altered Expression of βII- and βIII-Tubulin Isotypes

Peloruside A and laulimalide are potent microtubule-stabilizing natural products with a mechanism of action similar to that of paclitaxel. However, the binding site of peloruside A and laulimalide on tubulin remains poorly understood. Drug resistance in anticancer treatment is a serious problem. We developed peloruside A- and laulimalide-resistant cell lines by selecting 1A9 human ovarian carcinoma cells that were able to grow in the presence of one of these agents. The 1A9-laulimalide resistant cells (L4) were 39-fold resistant to the selecting agent and 39-fold cross-resistant to peloruside A, whereas the 1A9-peloruside A resistant cells (R1) were 6-fold resistant to the selecting agent while they remained sensitive to laulimalide. Neither cell line showed resistance to paclitaxel or other drugs that bind to the taxoid site on β-tubulin nor was there resistance to microtubule-destabilizing drugs. The resistant cells exhibited impaired peloruside A/laulimalide-induced tubulin polymerization and impaired mitotic arrest. Tubulin mutations were found in the βI-tubulin isotype, R306H or R306C for L4 and A296T for R1 cells. This is the first cell-based evidence to support a β-tubulin-binding site for peloruside A and laulimalide. To determine whether the different resistance phenotypes of the cells were attributable to any other tubulin alterations, the β-tubulin isotype composition of the cells was examined. Increased expression of βII- and βIII-tubulin was observed in L4 cells only. These results provide insight into how alterations in tubulin lead to unique resistance profiles for two drugs, peloruside A and laulimalide, that have a similar mode of action.

Specific β-Tubulin Isotypes Can Functionally Enhance or Diminish Epothilone B Sensitivity in Non-Small Cell Lung Cancer Cells

Epothilones are a new class of microtubule stabilizing agents with promising preclinical and clinical activity. Their cellular target is β-tubulin and factors influencing intrinsic sensitivity to epothilones are not well understood. In this study, the functional significance of specific β-tubulin isotypes in intrinsic sensitivity to epothilone B was investigated using siRNA gene knockdown against βII-, βIII- or βIVb-tubulins in two independent non-small cell lung cancer (NSCLC) cell lines, NCI-H460 and Calu-6. Drug-treated clonogenic assays showed that sensitivity to epothilone B was not altered following knockdown of βII-tubulin in both NSCLC cell lines. In contrast, knockdown of βIII-tubulin significantly increased sensitivity to epothilone B. Interestingly, βIVb-tubulin knockdowns were significantly less sensitive to epothilone B, compared to mock- and control siRNA cells. Cell cycle analysis of βIII-tubulin knockdown cells showed a higher percentage of cell death with epothilone B concentrations as low as 0.5 nM. In contrast, βIVb-tubulin knockdown cells displayed a decrease in epothilone B-induced G2-M cell cycle accumulation compared to control siRNA cells. Importantly, βIII-tubulin knockdowns displayed a significant dose-dependent increase in the percentage of apoptotic cells upon treatment with epothilone B, as detected using caspase 3/7 activity and Annexin-V staining. Higher concentrations of epothilone B were required to induce apoptosis in the βIVb-tubulin knockdowns compared to control siRNA, highlighting a potential mechanism underlying decreased sensitivity to this agent. This study demonstrates that specific β-tubulin isotypes can influence sensitivity to epothilone B and may influence differential sensitivity to this promising new agent.

Measuring the persistence length of MCF7 cell microtubules in vitro

The dynamic and mechanical properties of mammalian neural microtubules have been widely studied; however, similar knowledge about these properties is limited for non-neural microtubules, which, unlike neural microtubules, consist of different β-tubulin isotypes. In this study, we report, for the first time, an estimated value for the persistence length of a single non-neural microtubule polymerized from purified tubulin from human breast cancer cell lines (MCF7 tubulin). The method of measurement is based on an analysis of the local curvature of a microtubule as a result of thermal fluctuations. In parallel, we measured the persistence length of a single bovine brain microtubule under similar conditions. The results of our measurements indicate a higher value for the persistence length of MCF7 microtubules in vitro as compared to the persistence length of a neural microtubule. The difference can be associated with different β-tubulin isotypes in the structure of MCF7 microtubules.

Intraflagellar transport delivers tubulin isotypes to sensory cilium middle and distal segments

SUMMARYSensory cilia are assembled and maintained by kinesin-2-dependent intraflagellar transport (IFT). We investigated if two C. elegans α- and β-tubulin isotypes, identified via mutants that lack their cilium distal segments, are delivered to their assembly sites by IFT. Mutations in conserved residues in both tubulins destabilize distal singlet microtubules (MTs). One isotype, TBB-4, assembles into MTs at the tips of the axoneme core and distal segments, where the MT tip-tracker, EB1, is found, and localizes all along the cilium, whereas the other, TBA-5, concentrates in distal singlets. IFT assays, FRAP analysis and modeling suggest that the continual transport of sub-stoichiometric numbers of these tubulin subunits by the IFT machinery can maintain sensory cilia at their steady state length.

Regulation of β‐tubulin isotypes by micro‐RNA 100 in MCF7 breast cancer cells

Antimitotic drugs are key components of combination chemotherapy protocols for hematological and solid tumors. The taxanes (e.g., paclitaxel) bind to the β subunit of the tubulin heterodimer and reduce microtubule dynamics, leading to cell cycle arrest in G2/M. The effectiveness of combination chemotherapy is limited by tumor resistance to drugs initially or as a cumulative effect after several cycles of treatment. Because changes in the drug receptor may be linked to drug resistance, we investigated changes in β-tubulin isotypes in response to paclitaxel treatment in MCF7 breast cancer cells. We found that paclitaxel induced a 2-3 fold increase in mRNA for β-tubulin IIA and III genes, TUBB2A, and TUBB3. β-Tubulin class III protein increased; however, β-tubulin class II protein was not detected in these cells. Paclitaxel treatment following pretreatment with actinomycin D showed that the change in β-tubulin class III was due to increased transcription and linked to G2/M arrest. The increase in β-tubulin IIA mRNA was due to both enhanced stability and increased transcription, unassociated with G2/M arrest. We used micro-RNA superarrays to look for changes in families of micro-RNAs that might be linked to drug-induced changes in β-tubulin isotype mRNA and/or protein. We found a significant decrease in the tumor suppressor, miR-100, in MCF7 cells in response to paclitaxel treatment. Transfection of MCF7 cells with miR-100 significantly reduced β-tubulin I, IIA, IIB and V mRNA and prevented paclitaxel-induced increases in β-tubulin isotypes. This is the first report of a micro-RNA that regulates these specific β-tubulin isotype mRNAs.

β-Tubulin genotypes in six species of cyathostomins from anthelmintic-naive Przewalski and benzimidazole-resistant brood horses in Ukraine

Resistance to benzimidazoles (BZ) in the gastrointestinal nematodes of livestock is characterised by the presence of specific polymorphisms in the β-tubulin isotype 1 protein, a component of microtubules. The most prevalent polymorphism associated with resistance in nematodes infecting cattle, sheep, and goats is found at codon 200, with minor occurrences of polymorphisms at codons 167 and 198. In the cyathostomins that infect horses, however, a polymorphism at codon 167 appears to be more common than the codon 200 polymorphism. In the present study, a focussed analysis of PCR-amplified β-tubulin fragments incorporating the above-mentioned three polymorphic sites in isotype 1 and 2 genes in worms of six species of cyathostomins, Cylicocyclus nassatus, Cylicocyclus ashworthi, Cyathostomum catinatum, Cylicostephanus goldi, Cylicostephanus longibursatus, and Coronocyclus coronatus, was performed. Worms were collected from two distinct horse populations, i.e. they were collected from Przewalski horses of the Askania-Nova Biosphere Reserve that had never received any anthelmintic treatment and from brood horses of the Dubrovsky farm where benzimidazole resistance had become established. DNA was extracted and sequenced from three worms of each species and population as well as from pools of 50 male C. nassatus and C. catinatum from both populations. The vast majority of putatively BZ resistance-associated TAC alleles were found at codon 167, compared to codon 200. The former allele occurred in isotype 1 in all six species of the supposedly benzimidazole-resistant cyathostomins from Dubrovsky horses. None of the polymorphisms associated with resistance was found in the corresponding isotype 2 codons nor at codon 198 in any of the six species of cyathostomins (neither single nor pooled worm DNA) from either of the two populations. These findings further support the predominant association of β-tubulin isotype 1 and therein codon 167 with BZ resistance in cyathostomins.

Abstract 664: Polymerization of human βIII-tubulin is distinct from βI-tubulin in a cell-free system

Abstract Tubulins are a family of proteins consisting of 7 α- and 6 β-tubulin isotypes. Many studies have demonstrated that tumors overexpressing βIII-tubulin have a poor response to chemotherapy. Suppression of βIII-tubulin sensitizes non-small cell lung cancer (NSCLC) cells to microtubule-interacting agents, such as Taxol. βIII-tubulin has also been shown to mediate the incidence and progression of lung cancer. Tubulin isotype content of βI-, βIII-, βIV- and βV-tubulin in the human lung carcinoma cell line A549 is 50%, 8%, 36.5%, and 5.5%, respectively. In our study, the ability of six microtubule stabilizing agents, Taxol, epothilone B (EpoB), discodermolide (Disco), Ixabepilone, laulimalide and peloruside A, to polymerize tubulin in a 100,000 × g supernatant prepared from A549 cells was examined. The level of different tubulin isotypes in the drug-polymerized tubulin was determined by Western blot analysis with tubulin isotype specific antibodies. In the absence of drug, the level of polymerized βI-, βIII-, βIV- and βV-tubulin was 55%, 9%, 25%, and 48%, respectively. Ten µM Taxol caused a smalll increase (<15%) in βI- and βV- tubulin polymerization, but it induced a 367% and 228% increase in βIII- and βIV-tubulin polymerization. Among the six drugs tested, Disco and EpoB exhibited the greatest ability to polymerize all isotypes. The differential βI- and βIII-tubulin polymerization effects also were observed when intact A549 cells were lysed in the presence of drugs, followed by centrifugation. In the absence of drug, the level of polymerized βI- and βIII-tubulin was 9% and 0.2%, respectively. In the presence of 10 µM Taxol, the level of polymerized βI- and βIII-tubulin was increased to 28% and 7% which corresponded to a 3- and 35-fold increase in βI- and βIII-tubulin polymerization, respectively. However, when purified bovine brain tubulin in the absence of all endogenous proteins was used, there was no differential effect of drugs on the polymerization of βI+II- and βIII-tubulin. The βI-, βII-, βIII-, and βIV-tubulin content in bovine brain is 3%, 58%, 25% and 14%, respectively. Disco and EpoB also have the strongest effects on the polymerization of all bovine brain tubulin isotypes. Our results indicate that polymerization of βIII-tubulin is distinct from that of βI-tubulin in a cell-free system and that endogenous cellular components may influence the polymerization of tubulin isotypes. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 664. doi:10.1158/1538-7445.AM2011-664

Abstract 4911: Molecular investigation of the FTI/Taxol synergy using an integrated framework of transcriptome analysis

Abstract Farnesyltransferase inhibitors (FTIs) are a class of anticancer drugs that have exhibited modest activity in the clinic as single agents, but were found to have promising activity in combination with the microtubule-stabilizing drug Taxol (TX), both in vitro and in vivo. Clinical trials with an FTI/TX combination have demonstrated a clinical benefit for a subset of patients refractory to previous taxane-treatment. However, the precise cellular machinery underlying the FTI/TX synergistic drug combination still needs to be elucidated. To this end, we undertook an unbiased experimental/computational approach designed to identify the key cellular components that either contribute to or drive the therapeutic synergy of this drug combination. The approach combined (i) measurements of drug-mediated changes on tubulin biomarkers and overall survival; (ii) whole-genome transcriptome analysis using the combination index drug synergy algorithm; (iii) utilization of interaction and functional databases to identify pathway connections and (iv) a reverse-engineering forward simulation (REFS) approach to identify genes that drive synergy. Drug-induced transcriptome changes were examined using whole-genome Affymetrix HGU133 Plus 2 microarrays. Gene changes deemed significant using a fold change criterion, identified as synergistic with the combination index analysis, and their biological relevance was assessed by experimental validation by qPCR and siRNA studies. Also, the transcriptome changes unique to the LNF/TX combination were evaluated in the context of detailed molecular interaction maps and groups of biological function. Our analyses identified several major network hubs that include: (i) overexpression of the class II β-tubulin isotype TUBB2A and of several heat shock protein such as HSP40 and HSP70 and (ii) synergistic overexpression of cell cycle genes CDKN1A and MDM2 (iii) through REFS modeling, prosaposin and granulin were identified as drivers of drug synergy (iv) induction of the focal adhesion molecule paxillin (PXN). Interestingly, stable knockdown of protein farnesyltransferase mimicked the effects of FTI treatment and resulted in strong substratum attachment and migration defects suggesting that PXN could be regulated by farnesyltransferase and its pharmacologic inhibitors. Ultimately, we plan to check the expression levels of the key genes in tumor biopsies from patients enrolled in the Phase 1 FTI/Taxotere clinical trial and correlate their expression in pre- and post-treatment biopsies with clinical response to the combination chemotherapy. Herein, we demonstrate a combined experimental and computational approach that includes functional analysis, data-driven simulation and experimental validation, and its power to identify key cellular components, both known and novel, in the anticancer activity of drug combinations exhibiting therapeutic synergy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4911. doi:10.1158/1538-7445.AM2011-4911

Prognostic utility of β-tubulin isotype III and correlations with other molecular and clinicopathological variables in patients with early breast cancer: a translational Hellenic Cooperative Oncology Group (HeCOG) study

We evaluated the prognostic and predictive utility of β-tubulin isotype III (TUBB3) tumour gene transcription in early breast cancer patients enrolled in a randomised study. Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) was applied for assessment of TUBB3, ER, PgR, HER2 and MAPT messenger RNA and immunohistochemistry (IHC) for protein expression in 314 patients enrolled in trial HE10/97, evaluating epirubicin-alkylator adjuvant chemotherapy with or without paclitaxel. High TUBB3 mRNA status was associated with advanced T stage, high histological grade, low mRNA and protein levels of ER, PgR and MAPT, and high levels of HER2 (p < 0.001). At a median follow-up of 98 months, multivariate analysis showed high TUBB3 mRNA status to have prognostic significance for DFS (HR = 1.83, 95% CI 1.25-2.68, p = 0.002) and OS (HR = 1.71, 95% CI 1.03-2.83, p = 0.038), along with the number of involved axillary nodes, PgR mRNA status and tumour grade. TUBB3 mRNA levels did not predict benefit from inclusion of paclitaxel in adjuvant chemotherapy (test for interaction p = 0.96 for OS, p = 0.46 for DFS). Transcriptional activity of β-tubulin isotype III in early breast cancer is an adverse prognostic factor, though not a predictive one for taxane efficacy.

Megakaryocyte lineage‐specific class VI β‐tubulin suppresses microtubule dynamics, fragments microtubules, and blocks cell division

Class VI β-tubulin (β6) is the most divergent tubulin produced in mammals and is found only in platelets and mature megakaryocytes. To determine how this unique tubulin isotype affects microtubule assembly and organization, we expressed the cDNA in tissue culture cells under the control of a tetracycline regulated promoter. The β6 coassembled with other endogenous β-tubulin isotypes into a normal microtubule array; but once the cells entered mitosis it caused extensive fragmentation of the microtubules, disrupted the formation of the spindle apparatus, and allowed entry into G1 phase without cytokinesis to produce large multinucleated cells. The microtubule fragments persisted into subsequent cell cycles and accumulated around the membrane in a marginal band-like appearance. The persistence of the fragments could be traced to a pronounced suppression of microtubule dynamic instability. Impairment of centrosomal nucleation also contributed to the loss of a normal microtubule cytoskeleton. Incorporation of β6 allowed microtubules to resist the effects of colcemid and maytansine, but not vinblastine or paclitaxel; however, cellular resistance to colcemid or maytansine did not occur because expression of β6 prevented cell division. The results indicate that many of the morphological features of megakaryocyte differentiation can be recapitulated in non-hematopoietic cells by β6 expression and they provide a mechanistic basis for understanding these changes.

Phenotypic spectrum of the tubulin-related disorders and functional implications of disease-causing mutations

A spectrum of neurological disorders characterized by abnormal neuronal migration, differentiation, and axon guidance and maintenance have recently been attributed to missense and splice-site mutations in the genes that encode α-tubulin and β-tubulin isotypes TUBA1A, TUBA8, TUBB2B, and TUBB3, all of which putatively coassemble into neuronal microtubules. The resulting nervous system malformations can include different types of cortical malformations, defects in commissural fiber tracts, and degeneration of motor and sensory axons. Many clinical phenotypes and brain malformations are shared among the various mutations regardless of structural location and/or isotype, while others segregate with distinct amino acids or functional domains within tubulin. Collectively, these disorders provide novel paradigms for understanding the biological functions of microtubules and their core components in normal health and disease.

Mitochondrial Interactosome in Energy Metabolism in Healthy and Cancer Cells

In this study we investigated intracellular distribution of β-tubulin isotypes, expression of mitochondrial creatine kinase (MtCK) and their roles in Mitochondrial Interactosome (supercomplex formed by ATP synthasom, ANT, MtCK, VDAC-tubulin and other VDAC-linker proteins) in adult rat cardiomyocytes, in cancerous HL-1 cells of cardiac phenotype and several other cancer cells lines. Immunofluorescent and western blot analysis of adult cardiac muscle cells revealed the presence of all β-tubulin isotypes with the very regular localization of tubulin II(β2) corresponding to mitochondrial “crystal like” pattern. Antibodies against tubulin IV(β4) revealed characteristic staining of microtubular network. Polymerized transversal lines of tubulin III(β3) matched with Z-lines (alpha-actinin) fixing, probably, microtubular network to sarcomer structure. Tubulin I(β5) distribution was diffusely spotted and fragmentary polymerized. In cancer cardiac HL-1 and other cancer cell types MI remodelling is characterized by the absence of II(β2). Additionally, these cells do not contain tubulin IV(β4) isotype creating micro-tubular network in adult cardiomyocytes and have diffusely distributed tubulin I(β5) and III(β3) isotypes. Other characteristic of cancer cells was the absence of both sarcomeric and ubiquitous mitochondrial isoform of creatine kinase (MtCK). In cardiac cells respiration is controlled by creatine via activation of MtCK within MI. Knowing that the extremely low apparent Km for free ADP in cancer cells as well as in isolated mitochondria is associated with the lack of tubulin II(β2) isotype, we can assume that tubulin II(β2) can specifically control the voltage dependent anion channel (VDAC) selective permeability resulting in adenine nucleotides micro-compartmentation within the MI. The absence of II(β2) tubulin isotype and MtCK isoforms induce significant alterations of phenotype and regulatory mechanisms of cancer cells energy metabolism. By these mechanisms, all mitochondrial ATP can be captured to maintain activated glycolysis and lactate production, known as Warburg effect.

Modelling the microtubule: towards a better understanding of short-chain fatty acid molecular pharmacology

Systems biology combines experimental data with computational modelling to describe complex biological mechanisms and pathways. Short-chain fatty acids (SCFAs-chemopreventive compounds produced in the colon lumen) impair microtubule (MT) function in colon cancer cells by altering the relative expression of β-tubulin isotypes. The β-tubulin isotype composition along MT fibres is believed to contribute to a "tubulin code" defining which microtubule-associated proteins (MAPs) and kinesins are recruited and the arrangement of tubulin post-transcriptional modifications (PTMs) along the fibre, which in turn dictate many critical cellular functions. SCFAs drive acetylation of many proteins by virtue of being histone deacetylase inhibitors (HDACi's). Known acetyl-proteins include transcription factors and cytoplasmic cytoskeletal keratins as well as histones. Disruption of the MT cytoskeleton is a prime target of many cancer therapies including anti-microtubule drugs (AMD). This review focuses on SCFAs as HDACi's and how they might affect tubulin dynamics, modifications and isotypes. It discusses the evolution of mechanistic models that have helped improve understanding of tubulin-MT structure and dynamics and how to develop these models, combined with those describing transcription and the cell cycle, could provide hypotheses for how SCFAs disrupt cytoskeletal function. The review demonstrates how systems biology could offer potentially novel ideas for therapies in the prevention and treatment of cancers through the continued development and elaboration of such models.

Immune Thrombocytopenia (ITP) and Hβ-1 Tubulin: The Arg307His Substiution Is Over-Represented and Identifies Patients with More Severe Disease

AbstractAbstract 2525Platelet production and function are dependent on the presence of the hematopoietic-specific β-tubulin isotype Hβ1 (Class VI), whose expression is restricted to platelets and megakaryocytes, constituting 90% of total platelet β-tubulin. Among the eight human β-tubulin isotypes, Hβ1 is the only isotype for which frequent non-synonymous single nucleotide polymorphisms (SNPs) have been described. Little is known regarding the role these SNPs play in platelet production and function. In ITP, It is accepted that both accelerated platelet destruction and decreased platelet production are central to the disease process but the exact pathophysiologic mechanisms underlying individual patient variation are unknown. Likewise, little is understood regarding the diverse clinical manifestations of ITP—which range from minor to life-threatening bleeding, and even thrombosis—among patients who otherwise appear to have similar disease. The central role of platelets in ITP together with the platelet-specific expression of Hβ1 tubulin prompted us to investigate the potential role of Hβ1 SNPs in the pathophysiology and disease manifestations of ITP. We sequenced the coding region of Hβ1 gene using genomic DNA extracted from whole blood of 98 mostly-Caucasian ITP patients and 360 Caucasian controls. Our results showed that one of the 6 reported SNPs, namely 27795494G>A leading to the substitution of arginine for histidine at amino acid 307 (Arg307His), was overrepresented in the ITP patient population as compared to the controls. Specifically, the A allele was overrepresented in the ITP population (A allele frequency of 19% versus 16%; p=0.04). Importantly, the frequency of the homozygous A/A genotype was also significantly higher in the ITP population compared to the control population (7.1% versus 3.9%; p=0.05), while we did not find any changes in the frequencies of the heterozygote and wild-type genotypes. We retrospectively examined the disease characteristics of the different genotype populations in the ITP group; namely the homozygous (A/A) Hβ1 SNP versus the heterozygote (A/G) and homozygote wild-type (G/G) groups, which were combined after all analyses showed them to be similar, and are herein referred to as A/G+G/G. Our analysis showed no significant differences in gender, age, age at initial ITP diagnosis and time since initial diagnosis, between the A/A and A/G+G/G genotypes; there was also no difference in the incidence of concurrent autoimmune conditions between the groups. However, we found that the percentage of patients with platelet counts less than 30K/μL at disease presentation was significantly higher in the A/A genotype compared to the combined A/G+G/G genotype group (100% versus 55%, p=0.035). Furthermore, the total number of different treatment types for ITP within each group was significantly different, with A/A patients requiring a mean of 7.6 treatments (range 4–14), while the A/G+G/G patients required a mean of 5.4 treatments (range, 0–14) (p=0.03) over the course of their diseases. There were no significant differences between the groups with regard to responses to individual treatments (intravenous immune globulin, anti-D, rituximab, eltrombopag, and romiplostim). Taken together, these results suggest that alterations in Hβ1 tubulin play a patholphysiologic role in the development of ITP and that patients with the homozygous A/A genotype have more severe disease at presentation and more difficulty with maintenance of long-term disease control. Disclosures:Bussel:Amgen: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Research Funding; GlaxoSmithKline: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Research Funding; Ligand: Membership on an entity's Board of Directors or advisory committees, Research Funding; Shionogi: Membership on an entity's Board of Directors or advisory committees, Research Funding; Eisai: Membership on an entity's Board of Directors or advisory committees, Research Funding; Cangene: Research Funding; Genzyme: Research Funding; IgG of America: Research Funding; Immunomedics: Research Funding; Sysmex: Research Funding.

The miR-200 family controls -tubulin III expression and is associated with paclitaxel-based treatment response and progression-free survival in ovarian cancer patients

Ovarian cancer remains one of the leading causes of cancer deaths. Thus, new biomarkers predictive of response to the standard paclitaxel-carboplatin treatment are needed to improve chemotherapy strategies. MicroRNAs have the potential to modify drug outcomes. Based on this, we have demonstrated in this study that patients with a high expression of the miR-200 family show low levels of β-tubulin class III in ovarian carcinoma. In addition, we have established the clinical relevance of these microRNAs for ovarian cancer patients' treatment response and survival. In a well-characterized series of 72 ovarian carcinomas, the expressions of miR-141, miR-200a, miR-200b, miR-200c, and miR-429 were quantified by quantitative reverse transcription-PCR, and the protein content of β-tubulin isotypes I, II, and III was determined by immunohistochemistry. The relationship between these microRNAs, β-tubulin expression, response to paclitaxel-based treatment, progression-free survival (PFS) and overall survival was determined. While isotype I had constant high levels, protein expression of β-tubulins II and III was mutually exclusive. Low tumoral miR-200 expression was significantly associated with high β-tubulin III protein content (P values range, 0.047-<0.0001), and patients without complete response (CR) had lower miR-200c levels than patients with CR (hazard ratio (HR)=1.43, 95% confidence interval (CI)=1.02-1.99, P=0.037, multivariate analysis). Additionally, low miR-200 family expression had a trend toward poor PFS (HR>2.0, P values 0.051, 0.054, and 0.079 for miR-200c, miR-141, and miR-429 respectively, multivariate analysis). In conclusion, miR-200 family members affect the final β-tubulin III protein content of ovarian carcinomas. Furthermore, these microRNAs might constitute the biomarkers of response to paclitaxel-based treatments and relapse/progression of advanced stage ovarian carcinoma patients.

H-TBP: an approach based on intron-length polymorphism for the rapid isolation and characterization of the multiple members of the β-tubulin gene family in Camelina sativa (L.) Crantz

We have developed a new version of the cTBP (combinatorial tubulin-based polymorphism) method, a previously described approach based on intron-length polymorphism (ILP), to rapidly characterize the β-tubulin gene family of Camelina sativa (L.) Crantz, a plant species of importance for oil production but still largely unexplored at genomic level. The method, named h-TBP, allows the rapid cloning of the β-tubulin genomic sequences that encompass the two introns, invariantly present at fixed positions within the coding region of the vast majority of the plant species. The β-tubulin sequences cloned by h-TBP also comprise part of exon1 and exon3 and the whole sequence of exon2. The h-TBP method has then been used to isolate, clone and characterize the β-tubulin gene family of C. sativa, composed of at least 20 different β-tubulin isotypes, named CsTUB1 through CsTUB20. The relatively high number of β-tubulin genes has been further substantiated by Southern-blot analysis. Comparison of the β-tubulin exon sequences of C. sativa with those of Arabidopsis thaliana, the closest relative among crucifers, defines distinct groups of putative orthologous genes, identified by a UPMGA cluster analysis. Analysis of the C. sativa β-tubulin intron sequences reveals some molecular features that can provide the first hints for the understanding of intron plasticity and evolution. From a more immediate perspective, these data provide the first substantial contribution to the characterization of the largely unexplored genome of C. sativa, and the tools for assisting programmes of breeding and selection of the most productive plants.

Effect of TBCD and its regulatory interactor Arl2 on tubulin and microtubule integrity

Assembly of the α/β tubulin heterodimer requires the participation of a series of chaperone proteins (TBCA-E) that function downstream of the cytosolic chaperonin (CCT) as a heterodimer assembly machine. TBCD and TBCE are also capable of acting in a reverse reaction in which they disrupt native heterodimers. Homologs of TBCA-E exist in all eukaryotes, and the amino acid sequences of α- and β-tubulin isotypes are rigidly conserved among vertebrates. However, the efficiency with which TBCD effects tubulin disruption in vivo depends on its origin: bovine (but not human) TBCD efficiently destroys tubulin and microtubules upon overexpression in cultured cells. Here we show that recombinant bovine TBCD is produced in HeLa cells as a stoichiometric cocomplex with β-tubulin, consistent with its behavior in vitro and in vivo. In contrast, expression of human TBCD using the same host/vector system results in the generation of TBCD that is not complexed with β-tubulin. We show that recombinant human TBCD functions indistinguishably from its nonrecombinant bovine counterpart in in vitro CCT-driven folding reactions, in tubulin disruption reactions, and in tubulin GTPase activating protein assays in which TBCD and TBCC stimulate GTP hydrolysis by β-tubulin at a heterodimer concentration far below that required for polymerization into microtubules. We conclude that bovine and human TBCD have functionally identical roles in de novo tubulin heterodimer assembly, and show that the inability of human TBCD to disrupt microtubule integrity upon overexpression in vivo can be overcome by siRNA-mediated suppression of expression of the TBCD regulator Arl2 (ADP ribosylation factor-like protein).

Immunohistochemical Study of HER2 and TUBB3 Proteins in Extramammary Paget Disease

Metastatic extramammary Paget disease (EMPD) is a potentially fatal malignancy for which effective chemotherapy and good biomarkers are desirable for management. We investigated the status of human epidermal growth factor receptor (HER2) and neuronal β-tubulin isotype (class III β-tubulin; TUBB3), whose overexpression is a factor involved in resistance of tumor cells to taxane derivatives) in 32 patients with EMPD. HER2 status was evaluated by immunohistochemistry followed by fluorescence in situ hybridization, and TUBB3 status was evaluated by immunohistochemistry. On the basis of the US Food and Drug Administration-approved criteria, 20 (63%) of the 32 EMPD tumors were found to overexpress HER2. Positive immunoreactivity for TUBB3 was observed in 7 (22%) of the 32 patients. Although some clinicopathologic variables (nodule formation, depth of tumor cells, presence of lymph node metastasis, and serum carcinoembryonic antigen level) were significantly associated with disease outcome (P < 0.05), HER2 gain or aberrant TUBB3 expression showed no significant correlation. However, the higher incidence of HER2 gain and the relatively lower incidence of aberrant TUBB3 expression suggested that HER2-targeted immunotherapy combined with taxane derivatives is warranted for metastatic EMPD, and that HER2 and TUBB3 status might be a good biomarker for determining the most appropriate therapeutic modality.

Liver fluke β-tubulin isotype 2 binds albendazole and is thus a probable target of this drug

Albendazole is a benzimidazole drug which can be used to treat liver fluke (Fasciola hepatica) infections. Its mode of action is believed to be the inhibition of microtubule formation through binding to β-tubulin. However, F. hepatica expresses at least six different isotypes of β-tubulin, and this has confused, rather than clarified, understanding of the molecular mechanisms of benzimidazole drugs in this organism. Recombinant F. hepatica β-tubulin proteins were expressed in, and purified from, Escherichia coli. These proteins were then used in pull-down assays in which albendazole was covalently linked to Sepharose. β-Tubulin isotype 2 was pulled down in this assay, and this interaction could be reduced by adding competing albendazole. Molecular modelling of β-tubulin isotypes suggests that changes in the side change conformations of residue 200 in the putative albendazole binding site may be important in determining whether, or not, a particular isotype will bind to the drug. These results, together with previous work demonstrating that albendazole causes disruption of microtubules in the liver fluke, strongly suggest that β-tubulin isotype 2 is one of the targets of this drug.

BCIRG 001 Molecular Analysis: Prognostic Factors in Node-Positive Breast Cancer Patients Receiving Adjuvant Chemotherapy

There are currently no validated factors predictive of response to taxanes in patients with breast cancer. We analyzed specimens from patients included in the Breast Cancer International Research Group (BCIRG) 001 trial, a randomized study which showed the superiority of docetaxel/doxorubicin/cyclophosphamide over fluorouracil/doxorubicin/cyclophosphamide as adjuvant therapy for node-positive operable breast cancer in terms of disease-free survival (DFS) and overall survival (OS). Immunohistochemical assessment of biological markers included histologic grade, tumor size, estrogen and progesterone receptors, lymph node status, HER2, MUC1, Ki-67/MIB-1, p53, Bcl-2, Bax, Bcl-XL, BAG-1, beta-tubulin isotypes II, III and IV, tau protein, and detyrosinated alpha tubulin. Associations between selected parameters and survival were tested through univariate analyses, then completed with multivariate analyses and a bootstrap resampling technique. In univariate analysis histologic grade, tumor size, number of involved nodes, estrogen and progesterone receptor status, p53, Ki-67, tubulin III, and tau protein were associated both with DFS and with OS. In multivariate analysis estrogen and progesterone receptors, tumor size, number of involved nodes, and Ki-67 protein were associated both with DFS and with OS, whereas tau protein levels were correlated with DFS and tubulin III and P53 were correlated with OS. No interaction was observed between Ki-67 and treatment allocation. We conclude that the expression in primary tumors of Ki-67 and p53 protein, as well as of the microtubule-related parameters tau protein and tubulin III, are independent prognostic factors in patients receiving adjuvant chemotherapy for node-positive breast cancer but are not predictive of benefit from docetaxel-containing adjuvant chemotherapy.