Beta-tubulin Isotype Research Articles

Class III β-Tubulin Is Constitutively Coexpressed With Glial Fibrillary Acidic Protein and Nestin in Midgestational Human Fetal Astrocytes: Implications for Phenotypic Identity

Class III beta-tubulin isotype (betaIII-tubulin) is widely regarded as a neuronal marker in developmental neurobiology and stem cell research. To test the specificity of this marker protein, we determined its expression and distribution in primary cultures of glial fibrillary acidic protein (GFAP)-expressing astrocytes isolated from the cerebral hemispheres of 2 human fetuses at 18 to 20 weeks of gestation. Cells were maintained as monolayer cultures for 1 to 21 days without differentiation induction. By immunofluorescence microscopy, coexpression of betaIII-tubulin and GFAP was detected in cells at all time points but in spatially distinct patterns. The numbers of GFAP+ cells gradually decreased from Days 1 to 21 in vitro, whereas betaIII-tubulin immunoreactivity was present in 100% of cells at all time points. beta-III-tubulin mRNA and protein expression were demonstrated in cultured cells by reverse-transcriptase-polymerase chain reaction and immunoblotting, respectively. Glial fibrillary acidic protein+/beta-III-tubulin-positive cells coexpressed nestin and vimentin but lacked neurofilament proteins, CD133, and glutamate-aspartate transporter. Weak cytoplasmic staining was detected with antibodies against microtubule-associated protein 2 isoforms. Confocal microscopy, performed on autopsy brain samples of human fetuses at 16 to 20 gestational weeks, revealed widespread colocalization of GFAP and betaIII-tubulin in cells of the ventricular/subventricular zones and the cortical plate. Our results indicate that in the midgestational human brain, betaIII-tubulin is not neuron specific because it is constitutively expressed in GFAP+/nestin+ presumptive fetal astrocytes.

Preclinical efficacy spectrum and pharmacokinetics of ixabepilone

Ixabepilone, a semisynthetic analog of natural epothilone B, was developed for use in cancer treatment. This study extends previous findings regarding the efficacy of ixabepilone and its low susceptibility to tumor resistance mechanisms and describes the pharmacokinetics of this new antineoplastic agent. The cytotoxicity of ixabepilone was assessed in vitro in breast, lung, and colon tumor cell lines and in vivo in human xenografts in mice. Antitumor activities of ixabepilone and taxanes were compared in multidrug-resistant models in vivo. Differential drug uptake of ixabepilone and paclitaxel was assessed in a P-glycoprotein (P-gp)-resistant colon cancer model in vitro. The pharmacokinetic profile of ixabepilone was established in mice and humans. Ixabepilone demonstrated potent cytotoxicity in a broad range of human cancer cell lines in vitro and in a wide range of xenografts in vivo. Ixabepilone was *3-fold more potent than docetaxel in the paclitaxel-resistant Pat-21 xenograft model (resistant due to overexpression of betaIII-tubulin and a lack of betaII-tubulin). Ixabepilone activity against P-gp-overexpressing breast and colon cancer was confirmed in in vivo models. Cellular uptake of ixabepilone, but not paclitaxel, was established in a P-gp-overexpressing model. The pharmacokinetics of ixabepilone was characterized by rapid tissue distribution and extensive tissue binding. Cytotoxicity studies against a range of tumor types in vitro and in vivo demonstrate that ixabepilone has potent and broad-spectrum antineoplastic activity. This is accompanied by favorable pharmacokinetics. Ixabepilone has reduced susceptibility to resistance due to P-gp overexpression, tubulin mutations, and alterations in beta-tubulin isotype expression.

Fasciola hepatica expresses multiple α- and β-tubulin isotypes

We have identified five α-tubulin and six β-tubulin isotypes that are expressed in adult Fasciola hepatica. Amino acid sequence identities ranged between 72 and 95% for fluke α-tubulin and between 65 and 97% for β-tubulin isotypes. Nucleotide sequence identity ranged between 68–77% and 62–80%, respectively, for their coding sequences. Phylogenetic analysis indicated that two of the α-tubulins and two of the β-tubulins were distinctly divergent from the other trematode and nematode tubulin sequences described in this study, whereas the other isotypes segregated within the trematode clades. With regard to the proposed benzimidazole binding site on β-tubulin, three of the fluke isotypes had tyrosine at position 200 of β-tubulin, two had phenylalanine and one had leucine. All had phenylalanine at position 167 and glutamic acid at position 198. When isotype RT-PCR fragment sequences were compared between six individual flukes from the susceptible Cullompton isolate and from seven individual flukes from the two resistant isolates, Sligo and Oberon, these residues were conserved.

Genetic analysis of a relationship between macrocyclic lactone and benzimidazole anthelmintic selection on Haemonchus contortus

We have found that in a laboratory strain of the nematode parasite of ruminants, Haemonchus contortus, repeated ivermectin treatment, in vivo in sheep, selected against TTC, coding for Phe, and for TAC, coding for Tyr, at codon 200 in beta-tubulin isotype 1. This 200Tyr single nucleotide polymorphism (SNP) has been associated with benzimidazole anthelmintic resistance. In this study, we investigated the alpha-tubulin and beta-tubulin sequences of 17 different field and laboratory strains/isolates of H. contortus with known treatment history and status for susceptibility or resistance to macrocyclic lactone (ML) or benzimidazole anthelmintics. DNA (genomic or cDNA) from 10 to 32 male parasites was sequenced for alpha-tubulin or beta-tubulin for each strain/isolate and the frequency of coding SNPs was compared between the different strains/isolates with known treatment history and drug resistance status. We have analyzed alpha-tubulin and beta-tubulin isotypes 1 and 2 genes from the different H. contortus strains/isolates and found that repeated ivermectin or moxidectin (both ML anthelmintics) use changes the frequency of beta-tubulin isotype 1 alleles and selects for the following SNPs: either from TTC (Phe) to TAC (Tyr) at codon 200 or codon 167, or from GCA (Ala) to GAA (Glu) at codon 198. 200Tyr and 167Tyr were associated with GTT (Val) or CTC/CTT (Leu) at codon 368, whereas 200Phe or 167Phe were associated with either ATT (Ile) or GTT (Val) at codon 368 in beta-tubulin isotype 1. The SNPs 200Tyr, 167Tyr in beta-tubulin isotype 1 have been described to confer benzimidazole resistance in nematodes and recently 198Ala has also been associated with benzimidazole resistance. Although MLs exert their antiparasitic effect through binding to ligand-gated chloride channels, benzimidazoles interact with beta-tubulin. These genetic analyses indicate that there is a correlation between exposure and/or resistance to MLs and an increase in the frequency of the beta-tubulin alleles containing codons, which are determinant for benzimidazole resistance. ML use may predispose parasitic nematodes to benzimidazole resistance. This has major implications for parasite control programs dependent on MLs/benzimidazole rotations and on the use of ML/benzimidazole combination therapy, such as for human lymphatic filariasis and nematode control in ruminant livestock.

Distinct roles of neurofilament and tubulin gene expression in axonal growth.

Tubulin and the neurofilament (NF) proteins, which are major constituents of the axonal cytoskeleton, play distinct roles in longitudinal and radial growth of axons. The predominantly longitudinal growth of axons in developing neurons correlates with relatively high levels of expression for a particular tubulin gene, encoding the class II beta tubulin isotype, whereas levels of gene expression for two other beta tubulin isotypes (classes I and IV) are comparable in developing and maturing neurons. Gene expression for the 68 kDa NF protein (NF68) is low during longitudinal growth. Conversely, relatively high levels of gene expression for NF68 and low levels for class II beta tubulin in maturing neurons correlate with the radial growth of axons. The developmental pattern of gene expression is recapitulated during axonal regeneration. Expression of the class II beta tubulin isotype correlates with the outgrowth (elongation) of regenerating sprouts.

Expression of an intron-containing β-tubulin mRNA in catfish olfactory epithelium

Beta-tubulin genes code for very similar proteins, sharing extensive identity in amino acid sequence within and across species, each of which manifests characteristic patterns of cell and tissue expression. While searching for olfactory specific mRNAs in the channel catfish (Ictalurus punctatus), we isolated a novel beta-tubulin cDNA. In the putative ORF, 1298 nucleotides were 80-88% identical to cloned cDNAs from zebrafish to human for beta-tubulin isotype IVb. This ORF is interrupted by an insert of 111 nucleotides located between the regions corresponding to exons 2 and 3 in other species. This insert lacks similarity to any sequence in the NCBI databases. We showed that this novel cDNA fragment hybridizes specifically to catfish olfactory epithelium mRNA on Northern analysis. Here we demonstrate by in situ analysis of catfish olfactory epithelium that the expression of this mRNA is spatially restricted to the outer two-thirds of each olfactory lamella where olfactory receptor neurons reside. These results suggest that this nucleotide sequence is the result of incomplete RNA transcript processing. The growing awareness of the regulatory roles played by RNAs transcribed from intronic regions of genes suggests that this observation may have relevance to regulation of gene expression in olfactory tissue during development and axon targeting.

Class III β-Tubulin and γ-Tubulin are Co-expressed and Form Complexes in Human Glioblastoma Cells

We have previously shown that the neuronal-associated class III beta-tubulin isotype and the centrosome-associated gamma-tubulin are aberrantly expressed in astrocytic gliomas (Cell Motil Cytoskeleton 2003, 55:77-96; J Neuropathol Exp Neurol 2006, 65:455-467). Here we determined the expression, distribution and interaction of betaIII-tubulin and gamma-tubulin in diffuse-type astrocytic gliomas (grades II-IV) (n = 17) and the human glioblastoma cell line T98G. By immunohistochemistry and immunofluorescence microscopy, betaIII-tubulin and gamma-tubulin were co-distributed in anaplastic astrocytomas and glioblastomas and to a lesser extent, in low-grade diffuse astrocytomas (P < 0.05). In T98G glioblastoma cells betaIII-tubulin was associated with microtubules whereas gamma-tubulin exhibited striking diffuse cytoplasmic staining in addition to its expectant centrosome-associated pericentriolar distribution. Treatment with different anti-microtubule drugs revealed that betaIII-tubulin was not associated with insoluble gamma-tubulin aggregates. On the other hand, immunoprecipitation experiments unveiled that both tubulins formed complexes in soluble cytoplasmic pools, where substantial amounts of these proteins were located. We suggest that aberrant expression and interactions of betaIII-tubulin and gamma-tubulin may be linked to malignant changes in glial cells.

Expression of beta-tubulin isotypes in human primary ovarian carcinoma

Objective. Selective expression of beta-tubulin isotypes has been reported to be one of the important mechanisms of taxane resistance. The purpose of this study was to evaluate the immunohistochemical expression of beta-tubulin isotypes using clinical samples of ovarian carcinoma treated by taxanes and to examine whether the protein levels of each of the beta-tubulin isotypes were correlated with the clinical features. Experimental design. We examined tumor samples taken from 77 ovarian carcinoma patients (54 patients treated with a taxane-based regimen and 23 treated with a taxane-free regimen), for the intrinsic protein level of beta-tubulin isotype (classes I, II, III and IV) expression using immunohistochemistry, and we evaluated the correlation of this protein level with the clinical features. The expression levels were scored by the proportion and intensity of the immunoreactive tumor cells. Results. High protein levels of classes I and IV beta-tubulin, and very low protein levels of class II beta-tubulin, and intermediate protein levels of class III beta-tubulin expression were demonstrated in a total of 77 ovarian carcinomas. As for the samples taken from the 54 patients treated with the taxane-based regimen, 40 samples demonstrated undetectable levels of class II beta-tubulin protein. The class II beta-tubulin expression-absent group was significantly correlated with advanced stage ( p = 0.024) and with a short period of progression-free survival (log-rank test, p = 0.022). Multivariate analyses demonstrated that the only significant independent prognostic indicator of a short period of progression-free survival was advanced stage, although a high expression of class III beta-tubulin was also prone to be associated with a short period of progression-free survival, but not significantly so ( p = 0.081). No such correlations or propensities were demonstrated in the 23 patients treated with the taxane-free regimen. Conclusions. In cases of ovarian carcinoma treated by taxanes, high expression of class III beta-tubulin seems to be associated with earlier recurrence, which is believed likely to be resistant relapse. In addition, loss of class II beta-tubulin expression is correlated with advanced stage, which may represent aggressive tumor progression.

Expression of class II ?-tubulin in non-melanoma cutaneous tumors

Different combinations of beta-tubulin isotypes contribute to the diverse functions of microtubules (MTs). Class II beta-tubulin (class II tubulin) is up-regulated in differentiated keratinocytes. In contrast, the expression of class II tubulin in follicular differentiation and cutaneous tumors has not been studied. The immunohistochemical expression of class II tubulin was investigated in 117 cutaneous tumors: 30 squamous cell carcinomas (SCCs), seven keratoacanthomas (KAs), 57 basal cell carcinomas (BCCs), 23 trichoepitheliomas (TEs), and in the adjacent non-neoplastic skin. Class II tubulin was expressed in the keratinocytes of the granular layer, melanocytes, hair cortical and cuticular cells, inner root sheath (IRS), companion layer (CL) of the outer root sheath (ORS), and mesenchymal cells with Schwannian or myogenic differentiation. Moreover, class II tubulin expression was increased in the areas of squamous or follicular differentiation in cutaneous tumors. On grading the follicular differentiation or myofibroblastic response with anti-class II tubulin, TE showed follicular differentiation more frequently (p < 0.001) with less of a myofibroblastic response (p = 0.001) than BCC. Class II tubulin expression is closely related to squamous or follicular differentiation and may be helpful in distinguishing most SCCs from KAs and BCC from TE. However, it does not reliably distinguish well-differentiated, crateriform SCC from KA.

Innovative restriction site created PCR-RFLP for detection of benzimidazole resistance in Teladorsagia circumcincta

Benzimidazole compounds, especially albendazole, are the most commonly used anthelmintics for deworming of small ruminants in Iran. It is believed that the therapeutic effects of the benzimidazoles (BZs) come through their binding capacity to the beta-tubulin isotype 1. Substitution of phenylalanine to tyrosine at position 200 of this polypeptide confers resistance to BZs. Several investigators developed different biological- and molecular-based techniques to demonstrate the occurrence of resistance in helminthes against BZs. To address the determination of resistance at position 200 of beta-tubulin isotype 1, we developed an innovative restriction site created polymerase chain reaction-restriction fragment length polymorphism, in which nucleotide A at the position of 637 upstream flanked by the first two coding sequences of the phenylalanine (TT) triplet was substituted through the nucleotide G. The introduced modification in forward primer (UTvet MF-primer) leads to the creation of restriction site (AACGTT) for PSP1. Therefore, in the case of normal allele only, PSP1 can cut the corresponding PCR product. In the first step, the genomic DNA was isolated from each single Teladorsagia circumcincta collected either from the abomasa of untreated (n = 35) or of 5 mg/kg BW 2.5% albendazole suspension-treated (n = 40) sheep. It was amplified with the primer pair, creating PCR product of 403 bp in length. In the second step, the PCR product was extracted from agarose gel and amplified with the modified forward primer (UTvet MF-primer) and the same reverse primer as in step 1, creating a PCR product of 222 bp. The PCR product was then cut with PSP1 to obtain in the case of normal allele two DNA products (183 and 39 bp). Eight of the 35 worms collected from the untreated sheep were BZ(SS) homozygotes, and the rest (27) were BZ(RS) heterozygotes. In our preliminary experiment, we could not find a BZ(RR) homozygote form within the examined samples. Five out of 40 worms collected from the albendazole-treated sheep were BZ(RR) homozygotes, whereas the rest (35) were BZ(RS) heterozygotes. No BZ(SS) homozygote form was detected within this group.

Class III β-tubulin is a marker of paclitaxel resistance in carcinomas of unknown primary site

In this study, we determine the prevalence and the prognostic value of the class III beta-tubulin microtubule protein examined immunohistochemically, in tumors of 40 patients with carcinomas of unknown primary site treated with paclitaxel-based chemotherapy. Immunohistochemical intensity of staining and percentage of cells were quantified. Clinical characteristics, response to chemotherapy, progression-free survival, and overall survival were assessed for relationships with the expression of class III beta-tubulin. The response rate was 17.9% (seven partial responses among 39 valuable patients), while eleven patients had a stable disease (28.2%) and 21 patients progressed on therapy (53.8%). Patients with high class III beta-tubulin expression were more resistant to taxane-based chemotherapy, defined as progression under treatment, while patient characteristics were not found to be correlated with response to chemotherapy. Patients whose tumors expressed high levels of class III beta-tubulin isotype had shorter overall survival, while there was a trend for an association with progression free survival. Multivariate analysis showed that class III beta-tubulin expression was independently correlated with progression free survival and overall survival. These findings suggest that a high level of expression of class III beta-tubulin in tumor cells is associated with resistance to paclitaxel and decreased survival in patients with carcinomas of unknown primary receiving paclitaxel-based chemotherapy.

A phase II study of a novel anti-tubulin, E7389, in patients with advanced non-small cell lung cancer (NSCLC)

7106 Background: E7389, a synthetic analog of halichondrin B that was isolated from a marine sponge, has broad anti-proliferative activity at nanomolar levels and a unique profile of tubulin interactions. Methods: This is an open-label, single-arm, stratified phase II study of E7389 in patients with measurable, recurrent and/or metastatic NSCLC who progressed during or after platinum-based doublet chemotherapy. E7389 (1.4 mg/m2) was administered as a bolus IV on days 1, 8, and 15 of a 28-day cycle to 72 patients (cohort 1) in stratum I (55 taxane pretreated patients) and stratum II (17 taxane-naive patients) and on Days 1 and 8 of a 21-day cycle (cohort 2), providing an additional 22 patients in stratum I. The primary efficacy endpoint was objective response rate to E7389 monotherapy. Results: As of 9 December 2005, 94 evaluable patients received E7389. Nineteen tumors were classified as squamous cell carcinomas, 39 as adenocarcinomas, and 36 were large cell carcinomas or unclassified. The median number of cycles completed was 3. Fifteen patients completed 6 or more cycles and 75 patients underwent tumor assessments after cycle 2. Major toxicities related to study drug included myelosuppression, nausea, fatigue, dehydration, arthralgias, dyspnea, and peripheral neuropathy. Based on RECIST criteria, 6 partial responses (PR) were observed among 94 evaluable patients (PR rate = 6.4%, 95% CI: 2.8%, 12.8%). For 33 patients the best response was stable disease (SD rate = 35.1%, 95% CI: 25.5%, 45.1%). Disease control rate (PR + SD) was 41.5% (95% CI: 31.4%, 51.7%). For cohort 1, the 12-week progression free survival rate was 57.2%. As of 9 December 2005, median PFS time was 108 days (95% CI = 55, min-max = 1–239+). Cohort 2 is being followed to estimate their 12-week PFS. The correlation of beta tubulin isotype, stathmin, microtubule-associated protein 4 (MAP4) and tau protein mRNA expression with tumor responses is on-going. Conclusions: Based on this data, E7389 has been shown to be safe and effective in the treatment of NSCLC patients. Updated information and results of molecular correlations of responses will be presented. [Table: see text]

Expression of recombinant β-tubulin alleles from Cylicocyclus nassatus (Cyathostominae)

Small strongyles (Cyathostominae) are common nematode parasites of horses that have developed resistance to the benzimidazole anthelmintics used to control their populations. Evidence suggests that the principal mechanism of resistance involves a phenylalanine-to-tyrosine mutation at codon 200 in the beta-tubulin proteins that are components of microtubules. Other works, however, suggest that a phenylalanine-to-tyrosine mutation at codon 167, or alternative mechanisms, may be involved. As part of an ongoing project examining the role that these two beta-tubulin mutations may play in benzimidazole resistance, we have cloned the wild-type allele and the two alleles with the phenylalanine-to-tyrosine mutations at codons 167 and 200 of the beta-tubulin isotype 1 gene from the small strongyle Cylicocyclus nassatus. In this work, we describe the construction of expression vectors containing these alleles and their expression in Escherichia coli.

Comparison of β‐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.

Detection of Human βV-Tubulin Expression in Epithelial Cancer Cell Lines by Tubulin Proteomics

Tubulin, the constitutive protein of microtubules, is a heterodimeric protein with an alpha and beta subunit, encoded in vertebrates by six and seven different genes, respectively. Each tubulin isotype can be identified by its divergent C-terminal sequence. Nevertheless, two groups of beta-tubulin isotypes can be distinguished by sequence alignment; one includes betaI-, betaII-, betaIVa-, and betaIVb-tubulin, and the other includes betaIII-, betaV-, and betaVI-tubulin. betaIII-tubulin overexpression has been associated with microtubule destabilization and resistance to Taxol. Recent data indicate that mouse betaV-tubulin overexpression in CHO cells results in profound microtubule disorganization and dependence of cells on Taxol for growth. Mouse and human betaV-tubulin sequences display several differences, such as their respective extreme C-terminus, suggesting that they may have different effects on microtubule stability and different affinities for drugs. When high-resolution isoelectric focusing, in-gel CNBr cleavage, and mass spectrometry were combined, we detected for the first time the betaV-tubulin protein in human cell lines and found that it was highly expressed in Hey, an epithelial ovarian cancer cell line. Our data confirm that human and rodent betaV-tubulins are distinct and indicate that, regardless of species, betaIII- and betaV-tubulin may be expressed in a complementary pattern at the protein level. Therefore, both betaIII- and betaV-tubulin expression levels should be systematically determined to assess the role of differential tubulin isotype expression in the response of tumors to drugs targeting microtubules.

Alterations of β‐tubulin isotypes in breast cancer cells resistant to docetaxel

Docetaxel is one of the most active drugs used to treat breast cancer. The cellular target of docetaxel is the microtubule, specifically the beta-tubulin subunit, that comprises a series of isotypes and that can modulate function. This study has examined the role of alteration in beta-tubulin isotypes in vitro and has sequenced the beta-tubulin gene to determine if there were mutations, both of which may represent important mechanisms of acquired resistance to docetaxel. Breast cancer cells, MCF-7 (oestrogen-receptor positive) and MDA-MB-231, (oestrogen-receptor negative) were made resistant to docetaxel in vitro. Expression of beta-tubulin isotypes (class I, II, III, IVa, IVb, and VI) was determined at the RNA and protein level using RT-PCR and western analysis, respectively. DNA sequencing evaluated the beta-tubulin gene. At the mRNA level, class I, II, III, and IVa beta-tubulin mRNA isotypes were over-expressed in docetaxel-resistant MCF-7 cells when compared with the docetaxel-sensitive parental cells. However, class VI beta-tubulin mRNA isotype expression was decreased in resistant cells. In MDA-MB-231 cells, there was a decrease in expression of the class I and class IVa beta-tubulin mRNA. However, there were increased expressions in class II, IVb, and VI beta-tubulin mRNA isotypes in resistant cells. Western analysis has confirmed corresponding increases in beta-tubulin protein levels in MCF-7 cells. However, in MDA-MB-231 cells, there were decreased protein levels for class II and class III beta-tubulin. This study demonstrates that altered expression of mRNA beta-tubulin isotypes and modulation of beta-tubulin protein levels are associated with acquired docetaxel resistance in breast cancer cells. This allows further understanding and elucidation of mechanisms involved in resistance to docetaxel.

Estrogen effects on tubulin expression and taxane mediated cytotoxicity in prostate cancer cells

The present study was designed to determine if estrogens change microtubule polymerization and modulate cell cycle progression in vitro, related to modulation of tubulin expression and to determine if estrogens had antagonistic or synergistic effects with microtubule active agents. cDNA array analysis of LNCaP cells treated with the estrogens, estradiol, estrone, diethylstilbestrol (DES), and 2-methoxyestradiol (2-ME) was carried out and the results confirmed by PCR and Western blotting. Microtubule arrays in cells treated with estrogens were assessed using indirect immunofluorescence. The effects of combining estrogens with taxane was assessed by MTT assay and flow cytometry for cell cycle kinetics. Human prostate cancer xenografts were treated with DES and docetaxel to assess the effects of combining estrogens and taxane in vivo. Treatment of LNCaP cells with DES and 2-ME suppressed transcripts and protein for beta-tubulin isotype IVa. This effect on tubulin synthesis was not blocked by estrogen or androgen receptor modulators. Other estrogens had no effect on beta-tubulin expression. 2-ME and DES decreased the density of microtubules. The administration of DES or 2-ME with paclitaxel enhanced cytotoxicity and G(2)-M arrest in vitro. DES enhanced tumor suppression in a human prostate cancer xenograft model when combined with the taxane docetaxel. The use of DES and 2-ME enhances the effects of taxanes and may be a novel and important means of increasing therapeutic efficacy of cytotoxic chemotherapy against prostate carcinoma.

Isolation and characterisation of a beta tubulin isotype 2 gene from two species of cyathostomin

This study describes the isolation and characterisation of beta tubulin isotype 2 cDNA sequences from two common species of cyathostomin, Cylicocyclus nassatus and Cyathostomum catinatum. The full-length cDNAs for these species were 1709 and 1753 bp in length, respectively, including 1350 bp of sequence inferred to encode 450 amino acids of peptide sequence. They had greatest identity with previously characterised isotype 2 sequences from Trichostrongylus colubriformis, Cooperia oncophora and Haemonchus contortus (96% for C. nassatus and 95% for C. catinatum), and grouped together with these sequences in phylogenetic analysis. Both cyathostomin beta tubulin isotype 2 sequences contained the isotype-specific carboxyl terminal region described previously in other nematode species. Alignment with beta tubulin isotype 1 proteins from other trichostrongyloids, indicated 95 and 94% identity for the isotype 2 sequences of C. nassatus and C. catinatum, respectively. This comparison revealed 14 isotype-specific amino acid substitutions. Also, 2605 bp of beta tubulin isotype 2 genomic DNA sequence were isolated from C. nassatus. Comparison with the previously published isotype 1 gene of C. nassatus indicated differences in genomic organisation between the two isotypes. Reverse transcriptase (RT)-polymerase chain reaction analysis revealed constitutive temporal expression of beta tubulin isotype 1, whilst isotype 2 appeared to be developmentally expressed, with transcripts detected only in RNA derived from adult parasites.

Immunohistochemical Distribution of Tubulin beta II in Human Normal and Neoplastic Tissues

Tubulin is the major constituent protein of microtubules. In mammals, there are seven beta-tubulins and six alpha-tubulins. Each beta-tubulin isotype has a unique tissue distribution. The purpose of this study was to describe the distribution of tubulin beta II in normal and neoplastic human tissues with immunohistochemical techniques. We obtained normal tissues from 33 cases (8 fetuses, 17 neonates, 3 children and 5 adults) and 121 samples of neoplastic tissue from surgical specimens or at autopsy. Immunohistochemical staining for tubulin beta II was performed using a monoclonal antibody, KNY379 developed in our laboratory. Tubulin beta II was detected in various normal tissues, particularly in fetal and neonatal tissues, such as the nervous system, pulmonary alveoli, bronchioles and bronchi, colon, pancreatic ducts and acini, renal convoluted tubuli, skin epidermis, body cavity mesothelial cells, smooth muscle and thymus. In the adult, broad expression was also observed; however, the immunoreactivity was weaker and the extent of its distribution decreased with age. In neoplastic tissues, tubulin beta II immunoreactivity was detected in various nervous system neoplasms and other neoplasms such as pancreatic solid cystic carcinoma, pleomorphic adenoma, Warthin's tumor, nephroblastoma, basal cell carcinoma and malignant mesothelioma. We conclude that our monoclonal antibody, KNY379, may be useful as a marker of nervous system neoplasm, pancreatic solid cystic carcinoma, pleomorphic adenoma, Warthin's tumor, nephroblastoma, basal cell carcinoma and malignant mesothelioma.

Neuronal precursor‐specific activity of a human doublecortin regulatory sequence

The doublecortin (DCX) gene encodes a 40-kDa microtubule-associated protein specifically expressed in neuronal precursors of the developing and adult CNS. Due to its specific expression pattern, attention was drawn to DCX as a marker for neuronal precursors and neurogenesis, thereby underscoring the importance of its promoter identification and promoter analysis. Here, we analysed the human DCX regulatory sequence and confined it to a 3.5-kb fragment upstream of the ATG start codon. We demonstrate by transient transfection experiments that this fragment is sufficient and specific to drive expression of reporter genes in embryonic and adult neuronal precursors. The activity of this regulatory fragment overlapped with the expression of endogenous DCX and with the young neuronal markers class III beta-tubulin isotype and microtubule-associated protein Map2ab but not with glial or oligodendroglial markers. Electrophysiological data further confirmed the immature neuronal nature of these cells. Deletions within the 3.5-kb region demonstrated the relevance of specific regions containing transcription factor-binding sites. Moreover, application of neurogenesis-related growth factors in the neuronal precursor cultures suggested the lack of direct signalling of these factors on the DCX promoter construct.