Beta-tubulin Isotype Research Articles

Abstract 115: An integrated framework of transcriptome analysis leads to mechanistic insights into the FTI/Taxol synergy

Abstract A novel class of anticancer agents, the farnesyltransferase inhibitors (FTIs), has exhibited modest activity in the clinic as single agents, but was found to have promising activity in combination with the microtubule-stabilizing drug Taxol (TX), both in vitro and in vivo models. Recent phase I and II oncology 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 better understood. To this end, we undertook an unbiased experimental/computational approach that combined (i) measurements of drug-mediated changes on tubulin biomarkers and overall survival; (ii) whole-genome transcriptome analysis; (iii) utilization of interaction and functional databases to identify functional and pathway connections, (iv) computational modeling and prediction based on an in silico model predictive of drug synergy. Drug-induced transcriptome changes were examined using whole-genome Affymetrix HGU133 Plus 2 microarrays. The raw data were analyzed using two global statistical analysis methods (ANOVA and SAM). Gene changes that passed both tests, using a fold change criterion, were deemed significant and subjected to experimental validation by qPCR and siRNA. The transcriptome changes that were 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) repression of the mitotic spindle-checkpoint kinase, polo-like kinase 1 (Plk1) which is also implicated in carcinogenesis and was shown to directly bind and phosphorylate α- and β-tubulins. (ii) overexpression of CDKN1A and MDM2, both of which are shown to be inversely regulated by Plk1; (iii) overexpression of the class II beta tubulin isotype TUBB2A and of several heat shock protein such as HSPA1A and DNAJB9 and (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 paxillin could be regulated by farnesyltransferase and its pharmacologic inhibitors. Functional studies to elucidate this mechanism are currently underway. In summary, 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. (Supported by NCI P50 CA128613 & P01 CA116676) Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 115.

Evidence of an Asymmetrical Endophenotype in Congenital Fibrosis of Extraocular Muscles Type 3 Resulting fromTUBB3Mutations

Orbital magnetic resonance imaging (MRI) was used to investigate the structural basis of motility abnormalities in congenital fibrosis of the extraocular muscles type 3 (CFEOM3), a disorder resulting from missense mutations in TUBB3, which encodes neuron-specific beta-tubulin isotype III. Ophthalmic examinations in 13 volunteers from four CFEOM3 pedigrees and normal control subjects, were correlated with TUBB3 mutation and MRI findings that demonstrated extraocular muscle (EOM) size, location, contractility, and innervation. Volunteers included clinically affected and clinically unaffected carriers of R262C and D417N TUBB3 amino acid substitutions and one unaffected, mutation-negative family member. Subjects with CFEOM3 frequently had asymmetrical blepharoptosis, limited vertical duction, variable ophthalmoplegia, exotropia, and paradoxical abduction in infraduction. MRI demonstrated variable, asymmetrical levator palpebrae superioris and superior rectus EOM atrophy that correlated with blepharoptosis, deficient supraduction, and small orbital motor nerves. Additional EOMs exhibited variable hypoplasia that correlated with duction deficit, but the superior oblique muscle was spared. Ophthalmoplegia occurred only when the subarachnoid width of CN3 was <1.9 mm. A-pattern exotropia was frequent, correlating with apparent lateral rectus (LR) muscle misinnervation by CN3. Optic nerve (ON) cross sections were subnormal, but rectus pulley locations were normal. CFEOM3 caused by TUBB3 R262C and D417N amino acid substitutions features abnormalities of EOM innervation and function that correlate with subarachnoid CN3 hypoplasia, occasional abducens nerve hypoplasia, and subclinical ON hypoplasia that can resemble CFEOM1. Clinical and MRI findings in CFEOM3 are more variable than those in CFEOM1 and are often asymmetrical. Apparent LR innervation by the inferior rectus motor nerve is an overlapping feature of Duane retraction syndrome and CFEOM1. These findings suggest that CFEOM3 is an asymmetrical, variably penetrant, congenital cranial dysinnervation disorder leading to secondary EOM atrophy.

Tumoral and tissue‐specific expression of the major human β‐tubulin isotypes

The beta-tubulins are microtubule components encoded by a multigene family, which produces slightly different proteins with complex expression patterns. Several widely used anticancer drugs base their activity on beta-tubulin binding, microtubule dynamics alteration, and cell division blockage. The expression of these drug targets in tumoral and normal cells could be of crucial importance for therapy outcome, unfortunately, the complex beta-tubulin expression patterns have been poorly characterized in human. In this study, we developed a quantitative RT-PCR technique that accurately determines the mRNA expression of the eight human beta-tubulin isotypes, encoding class I, IIa, IIb, III, IVa, IVb, V, and VI and applied it to 21 nontumoral tissues and 79 tumor samples belonging to seven cancer types. In the nontumoral tissues, we found that, overall, TUBB (I), TUBB2C (IVb), and TUBB6 (V) were ubiquitous, TUBB1(VI) was hematopoietic cell-specific, and TUBB2A (IIa), TUBB2B (IIb), TUBB3 (III), and TUBB4 (IVa) had high expression in brain; however, the contribution of the different isotypes to the total beta-tubulin content varied for each tissue and had a complex pattern. In tumoral tissues, most isotypes exhibited an altered expression in specific tumor types or related to tumoral characteristics. In general, TUBB3 showed a great increase in expression while TUBB6 expression was largely decreased in most tumors. Thus, normal tissues showed a complex beta-tubulin isotype distribution, which could contribute to the toxicity profile of the microtubule-binding drugs. In addition, the specific isotypes significantly altered in tumors might represent markers for drug response.

Characterization of the Colchicine Binding Site on Avian Tubulin Isotype βVI

Tubulin, the basic component of microtubules, is present in most eukaryotic cells as multiple gene products, called isotypes. The major tubulin isotypes are highly conserved in terms of structure and drug binding capabilities. Tubulin isotype betaVI, however, is significantly divergent from the other isotypes in sequence, assembly properties, and function. It is the major beta-tubulin isotype of hematopoietic tissue and forms the microtubules of platelet marginal bands. The interaction of the major tubulin isotypes betaI, betaII, betaIII, and betaIotaV with antimicrotubule drugs has been widely studied, but little is known about the drug binding properties of tubulin isotype betaVI. In this investigation, we characterize the activity of various colchicine site ligands with tubulin isolated from Gallus gallus erythrocytes (CeTb), which is approximately 95% betaVI. Colchicine binding is thought to be a universal property of higher eukaryotic tubulin; however, we were unable to detect colchicine binding to CeTb under any experimental conditions. Podophyllotoxin and nocodazole, other colchicine site ligands with divergent structures, were able to inhibit paclitaxel-induced CeTb assembly. Surprisingly, the colchicine isomer allocolchicine also inhibited CeTb assembly and displayed measurable, moderate affinity for CeTb (K(a) = 0.18 x 10(5) M(-1) vs 5.0 x 10(5) M(-1) for bovine brain tubulin). Since allocolchicine and colchicine differ in their C ring structures, the two C ring colchicine analogues were also tested for CeTb binding. Kinetic experiments indicate that thiocolchicine and chlorocolchicine bind to CeTb, but very slowly and with low affinity. Molecular modeling of CeTb identified five divergent amino acid residues within 6 A of the colchicine binding site compared to betaI, betaII, and betaIV; three of these amino acids are also altered in betaIII-tubulin. Interestingly, the altered amino acids are in the vicinity of the A ring region of the colchicine binding site rather than the C ring region. We propose that the amino acid differences in the binding site constrict the A ring binding domain in CeTb, which interferes with the positioning of the trimethoxyphenyl A ring and prevents C ring binding site interactions from efficiently occurring. Allocolchicine is able to accommodate the altered binding mode because of its smaller ring size and more flexible C ring substituents. The sequence of the colchicine binding domain of CeTb isotype betaVI is almost identical to that of its human hematopoietic counterpart. Thus, through analysis of the interactions of ligands with CeTb, it may be possible to discover colchicine site ligands that specifically target tubulin in human hematopoietic cells.

Microtubules and resistance to tubulin-binding agents

Microtubules are dynamic structures composed of alpha-beta-tubulin heterodimers that are essential in cell division and are important targets for cancer drugs. Mutations in beta-tubulin that affect microtubule polymer mass and/or drug binding are associated with resistance to tubulin-binding agents such as paclitaxel. The aberrant expression of specific beta-tubulin isotypes, in particular betaIII-tubulin, or of microtubule-regulating proteins is important clinically in tumour aggressiveness and resistance to chemotherapy. In addition, changes in actin regulation can also mediate resistance to tubulin-binding agents. Understanding the molecular mechanisms that mediate resistance to tubulin-binding agents will be vital to improve the efficacy of these agents.

Human TUBB3 Mutations Perturb Microtubule Dynamics, Kinesin Interactions, and Axon Guidance

We report that eight heterozygous missense mutations in TUBB3, encoding the neuron-specific beta-tubulin isotype III, result in a spectrum of human nervous system disorders that we now call the TUBB3 syndromes. Each mutation causes the ocular motility disorder CFEOM3, whereas some also result in intellectual and behavioral impairments, facial paralysis, and/or later-onset axonal sensorimotor polyneuropathy. Neuroimaging reveals a spectrum of abnormalities including hypoplasia of oculomotor nerves and dysgenesis of the corpus callosum, anterior commissure, and corticospinal tracts. A knock-in disease mouse model reveals axon guidance defects without evidence of cortical cell migration abnormalities. We show that the disease-associated mutations can impair tubulin heterodimer formation in vitro, although folded mutant heterodimers can still polymerize into microtubules. Modeling each mutation in yeast tubulin demonstrates that all alter dynamic instability whereas a subset disrupts the interaction of microtubules with kinesin motors. These findings demonstrate that normal TUBB3 is required for axon guidance and maintenance in mammals.

Tubulin targets in the pathobiology and therapy of glioblastoma multiforme. I. class III β‐tubulin

Glioblastoma multiforme (GBM) is the most common and deadliest form of primary brain cancer in adults. Despite advances in molecular biology and genetics of gliomas currently there is no effective treatment or promising molecularly targeted experimental therapeutic strategies for these tumors. In previous studies we have shown aberrant overexpression of the class III beta-tubulin isotype (betaIII-tubulin) in GBM and have proposed that this change may reflect perturbations in microtubule dynamics associated with glioma tumorigenesis, tumor progression and malignant transformation into GBM. This minireview focuses on microtubules and tubulin as emerging targets in potential therapy of GBM using a new class of betaIII-tubulin-targeted drugs in the light of recent developments concerning the function and potential role of this isotype in clinically aggressive tumor behavior, cancer stem cells, tumor hypoxia and chemoresistance to tubulin binding agents, principally taxanes.

Determination of genomic DNA sequences for beta-tubulin isotype 1 from multiple species of cyathostomin and detection of resistance alleles in third-stage larvae from horses with naturally acquired infections

BackgroundGenetic resistance against benzimidazole (BZ) anthelmintics is widespread in cyathostomins, the commonest group of intestinal parasitic nematodes of horses. Studies of BZ-resistant nematodes of sheep, particularly Haemonchus contortus, have indicated that an anthelmintic resistance-conferring T/A polymorphism, encoding an F (phenylalanine) to Y (tyrosine) substitution, in beta-tubulin isotype 1 is present at two loci, codons 167 and 200 (F167Y, F200Y). Recent studies using complementary (c) DNA derived from BZ-susceptible and -resistant cyathostomins identified statistical differences in the frequency of the BZ-resistant A allele at these loci. However, the lack of high-throughput genomic DNA-based detection of polymorphisms limits the study of eggs or larvae from field isolates. In the present study, we report genomic DNA sequences for beta-tubulin isotype 1 from multiple cyathostomin species, thus facilitating the development of pyrosequencing assays to genetically characterize third-stage larvae (L3s) of cyathostomins from mixed-species field isolates.ResultsSequence analysis of the beta-tubulin isotype 1 gene in a common species, Cylicocyclus nassatus, indicates a revised genomic structure to published data, revealing that codons 167 and 200 are located on separate exons. A consensus sequence was generated from 91 and 76 individual cyathostomins for the regions spanning codons 167 and 200, respectively. A multi-species genomic DNA-based assay was established to directly pyrosequence individual L3 from field samples of unknown species and BZ sensitivity in a 96-well plate. In this format, the assay to detect F167Y gave a 50-90% success rate. The optimisation of the assay at codon 200 is currently underway. Subsequently, the genotype at F167Y was determined for 241 L3s, collected prior to and after BZ treatment. These results demonstrated a reduction in the heterozygous genotype, TTC/TAC, and an increase in the homozygous resistant genotype TAC/TAC in post-treatment samples. However, the differences in allele frequencies determined before and after BZ treatment were not statistically significant.ConclusionExtensive genomic DNA sequence, spanning codons 167 and 200 of the beta-tubulin isotype 1 gene, was generated from multiple cyathostomin species. The data facilitated the development of a pyrosequencing assay, capable of detecting the genotype of individual cyathostomin L3s derived from mixed-species field samples. Differences in codon 167 allele frequencies were observed in L3s isolated pre- and post-BZ treatment.

Tubulin isotype specificity and identification of the epitope for antibody Tub 2.1

Tub 2.1, a monoclonal antibody commonly used to measure cellular tubulin content, is widely believed to recognize all beta-tubulin isotypes. Though it has been used for more than two decades, the epitope for this antibody is not well established. We report for the first time that contrary to common belief, this antibody does not react with all isotypes of beta-tubulin. Of the seven vertebrate beta-tubulins, the more divergent class V and VI isotypes are not recognized by this antibody. Among the isotypes that do react, binding is similar for beta2, beta3, beta4a and beta4b but lower for beta1, the most abundant isotype. Expression of chimeric tubulins verified that the epitope for Tub 2.1 is near the C-terminal end of beta-tubulin. Site-directed mutagenesis of this region in nonreactive beta5 to match the sequence of beta4b resulted in strong reaction to Tub 2.1 and narrowed the epitope to amino acids 431-436.

Stress hormones mediate drug resistance to paclitaxel in human breast cancer cells through a CDK-1-dependent pathway

Chemotherapy comprises part of successful treatment regimens for breast cancer, however, up to 50% of patients develop resistance. Stress in cancer patients can equate to poor chemotherapeutic responses. We hypothesize that drug resistance may be associated with stress hormone-induced alterations in breast cancer cells. To test this hypothesis, MDA-MB-231 cells were cultured with paclitaxel and/or cortisol, norepinephrine and epinephrine and cytotoxicity, cell cycle analyses, genomic and proteomic analyses were performed. Paclitaxel-mediated cytotoxicity and G2/M cell cycle arrest were reversed significantly by stress hormones. Genomic and proteomic analyses revealed that stress hormones modulated beta-tubulin isotypes and significantly altered genes and proteins involved in regulation of the G2/M transition, including cyclin-dependent kinase-1 (CDK-1). Inhibition of CDK-1 abrogated stress hormone-mediated reversal of paclitaxel-induced cytotoxicity, indicating that the protective effect of stress hormones act through a CDK-1-dependent mechanism. These data demonstrate that stress hormones interfere with paclitaxel efficacy and contribute significantly to drug resistance.

Identification of two ß-tubulin isotypes of Clonorchis sinensis

The beta-tubulin coding sequences were investigated for the Chinese liver fluke, Clonorchis sinensis. A cDNA library of adult C. sinensis was screened with cDNA probes synthesized by polymerase chain reaction using degenerate primers, and two beta-tubulin cDNAs, CsTB1 and CsTB3, were subsequently cloned. The CsTB1 and CsTB3 cDNA were 2,082 and 1,486 bp long and encoded 445 and 444 amino acids, respectively. The two clones were identical 78% in their coding sequences and 97% in deduced sequences. Moreover, the CsTB1 had 3'-UTR 592 bp longer than that of CsTB3. The CsTB1 and CsTB3 polypeptides were highly homologous (88-99%) with those of known beta-tubulins of other helminthes with a variation at the C-terminal region. In CsTB1 and CsTB3 polypeptides, two amino acids Tyr 200 and Phe 167 were conserved. The phylogenetic analysis of selected sequences indicates that the beta-tubulins can be organized into five groups and that the two beta-tubulin isotypes of C. sinensis are contained within one group with those of other trematodes and vertebrates.

Molecular Basis for Class V β-Tubulin Effects on Microtubule Assembly and Paclitaxel Resistance

Vertebrates produce at least seven distinct beta-tubulin isotypes that coassemble into all cellular microtubules. The functional differences among these tubulin isoforms are largely unknown, but recent studies indicate that tubulin composition can affect microtubule properties and cellular microtubule-dependent behavior. One of the isotypes whose incorporation causes the largest change in microtubule assembly is beta5-tubulin. Overexpression of this isotype can almost completely destroy the microtubule network, yet it appears to be required in smaller amounts for normal mitotic progression. Moderate levels of overexpression can also confer paclitaxel resistance. Experiments using chimeric constructs and site-directed mutagenesis now indicate that the hypervariable C-terminal region of beta5 plays no role in these phenotypes. Instead, we demonstrate that two residues found in beta5 (Ser-239 and Ser-365) are each sufficient to inhibit microtubule assembly and confer paclitaxel resistance when introduced into beta1-tubulin; yet the single mutation of residue Ser-239 in beta5 eliminates its ability to confer these phenotypes. Despite the high degree of conservation among beta-tubulin isotypes, mutations affecting residue 365 demonstrate that amino acid substitutions can be context sensitive; i.e. an amino acid change in one isotype will not necessarily produce the same phenotype when introduced into a different isotype. Modeling studies indicate that residue Cys-239 of beta1-tubulin is close to a highly conserved Cys-354 residue suggesting the possibility that disulfide formation could play a significant role in the stability of microtubules formed with beta1- but not with beta5-tubulin.

Roles of β-Tubulin Residues Ala428 and Thr429 in Microtubule Formation in Vivo

The C termini of beta-tubulin isotypes are regions of high sequence variability that bind to microtubule-associated proteins and motors and undergo various post-translational modifications such as polyglutamylation and polyglycylation. Crystallographic analyses have been unsuccessful in resolving tubulin C termini. Here, we used a stepwise approach to study the role of this region in microtubule assembly. We generated a series of truncation mutants of human betaI and betaIII tubulin. Transient transfection of HeLa cells with the mutants shows that mutants with deletions of up to 22 residues from betaIII and 16 from betaI can assemble normally. Interestingly, removal of the next residue (Ala(428)) results in a complete loss of microtubule formation without affecting dimer formation. C-terminal tail switching of human betaI and betaIII tubulin suggests that C-terminal tails are functionally equivalent. In short, residues outside of 1-429 of human beta-tubulins make no contribution to microtubule assembly. Ala(428), in the C-terminal sequence motif N-QQYQDA(428), lies at the end of helix H12 of beta-tubulin. We hypothesize that this residue is important for maintaining helix H12 structure. Deletion of Ala(428) may lead to unwinding of helix H12, resulting in tubulin dimers incapable of assembly. Thr(429) plays a more complex role. In the betaI isotype of tubulin, Thr(429) is not at all necessary for assembly; however, in the betaIII isotype, its presence strongly favors assembly. This result is consistent with a likely more complex function of betaIII as well as with the observation that evolutionary conservation is total for Ala(428) and frequent for Thr(429).

Identification and Characterization of an Intermediate Taxol Binding Site Within Microtubule Nanopores and a Mechanism for Tubulin Isotype Binding Selectivity

Tubulin, the primary subunit of microtubules, is remarkable for the variety of small molecules to which it binds. Many of these are very useful or promising agents in cancer chemotherapy. One of the most useful of these is paclitaxel. The tubulin molecule is itself an alpha/beta heterodimer, both alpha- and beta-tubulin monomers existing as multiple isotypes. Despite the success of paclitaxel as an anticancer drug, resistance often occurs in cancer cells and has been associated with variations in tubulin isotype expression, most notably with the increased expression of betaIII-tubulin. Paclitaxel is thought to reach its binding site on beta-tubulin by diffusion through nanopores in the microtubule wall. It has been suggested that a transitional step in this process may be the binding of paclitaxel to an intermediate site within a nanopore, from which it moves directly to its binding site in the microtubule interior facing the lumen. To test this hypothesis, we have computationally docked paclitaxel within a microtubule nanopore and simulated its passage to the intermediate binding site. Targeted molecular dynamics was then used to test the hypothesis that paclitaxel utilizes the H6/H7 loop as a hinge to move directly from this intermediate binding site to its final position in the luminal binding site. We observed that this motion appears to be stabilized by the formation of a hydrogen bond involving serine 275 in beta-tubulin isotypes I, IIa, IIb, IVa, IVb, V, VII, and VIII. Interestingly, this residue is replaced by alanine in the betaIII and VI isotypes. This observation raises the possibility that the observed isotype difference in paclitaxel binding may be a kinetic effect arising from the isotype difference at this residue. We are now able to suggest derivatives of paclitaxel that may reverse the isotype-specificity or lead to an alternate stabilizing hydrogen-bond interaction with tubulin, thus increasing the rate of passage to the luminal binding site and hopefully offering a therapeutic advantage in paclitaxel resistant cases.

Chemically defined sequential culture media for TH+ cell derivation from human embryonic stem cells

During the past few years several differentiation protocols to derive midbrain dopamine (DA) neurons from human embryonic stem (hES) cells have been developed, but the production of sufficient amounts of the 'right' therapeutic DA cells has not yet been accomplished. The aim of this study was to efficiently generate tyrosine hydroxylase (TH)-positive cells in vitro from our hES cells using a chemically defined culture system. At the end of differentiation, the vast majority of cells (>90%) were positive for both TH and beta-tubulin isotype III (TuJ1). Other markers of dopaminergic cells, like dopamine transporter (DAT) and Nurr1 were also detected by immunofluorescence or RT-PCR. The functions of these cells were confirmed by measurements of DA release in vitro and by transplantation of derived cells into Parkinson's disease (PD) rats in vivo. We found these cells were able to release DA when depolarized by high K(+). Moreover, 4 weeks after transplantation, the hES-derived cells could survive and reduce the apomorphine-induced rotation behaviour of the rats. In conclusion, the experimental system presented here provided a reliable protocol to produce a large number of hES-derived TH(+) cells which may be used in cell therapy for PD in future.

β‐tubulin isotype classes II and V expression patterns in nonsmall cell lung carcinomas

Previous studies suggest that beta-tubulin isotype protein levels could be useful as indicators of nonsmall cell lung cancer (NSCLC) aggressiveness. However, measurement of protein amounts in tissue samples by staining techniques is semiquantitative at best. Since technologies for measuring mRNA levels have become more efficient and quantitative, we wanted to determine whether beta-tubulin message levels may be useful as biomarkers. Quantitative real-time RT-PCR was used to measure the seven classes of beta-tubulin isotypes, stathmin and MAP4 mRNA levels in 64 NSCLC and 12 normal lung tissue samples. We found significantly higher fractions of beta-tubulin classes II and V mRNA compared to the other isotypes in all lung tumor samples (P < 0.05). In addition, the ratio of beta-tubulin classes II/V mRNA was significantly higher in NSCLCs compared to normal lung tissues (P < 0.001). The data suggest that the ratio of beta-tubulin classes II and V mRNA could be useful as a biomarker for NSCLC tumor differentiation and/or NSCLC aggressiveness. Furthermore, the ratio of MAP4 to stathmin mRNA was found to be higher in diseased lung tissues compared to normal lung tissues, suggesting this ratio might also be used as a clinically relevant biomarker for NSCLCs.

Increased Levels of a Unique Post-Translationally Modified βIVb-Tubulin Isotype in Liver Cancer

Identifying changes at the molecular level during the development of hepatocellular carcinoma is important for the detection and treatment of the disease. The characteristic structural reorganization of preneoplastic cells may involve changes in the microtubule cytoskeleton. Microtubules are dynamic protein polymers that play an essential role in cell division, maintenance of cell shape, vesicle transport, and motility. They are comprised of multiple isotypes of alpha- and beta-tubulin. Changes in the levels of these isotypes may affect not only microtubule stability and sensitivity to drugs but also interactions with endogenous proteins. We employed a rat liver cancer model that progresses through stages similar to those of human liver cancer, including metastasis to the lung, to identify changes in the tubulin cytoskeleton during carcinogenesis. Tubulin isotypes in both liver and lung tissue were purified and subsequently separated by isoelectric focusing electrophoresis. The C-terminal isotype-defining region from each tubulin was obtained by cyanogen bromide cleavage and identified by mass spectrometry. A novel post-translational modification of betaIVb-tubulin in which two hydrophobic residues are proteolyzed from the C-terminus, thus exposing a charged glutamic acid residue, was identified. The unique form of betaIVb-tubulin was quantified in the liver tissue of all carcinoma stages and found to be approximately 3-fold more abundant in nodular and tumor tissue than in control tissue. The level of this form was also found to be increased in lung tissue with liver metastasis. This modification alters the C-terminal domain of one of the most abundant beta-tubulin isotypes in the liver and therefore may affect the interactions of microtubules with endogenous proteins.

Induced in vitro differentiation of neural-like cells from human amnion-derived fibroblast-like cells

There is growing evidence that the human amnion contains various types of stem cell. As amniotic tissue is readily available, it has the potential to be an important source of material for regenerative medicine. In this study, we evaluated the potential of human amnion-derived fibroblast-like (HADFIL) cells to differentiate into neural cells. Two HADFIL cell populations, derived from two different neonates, were analyzed. The expression of neural cell-specific genes was examined before and after in vitro induction of cellular differentiation. We found that neuron specific enolase, neurofilament-medium, beta-tubulin isotype III, and glial fibrillary acidic protein (GFAP) showed significantly increased expression following the induction of differentiation. In addition, immunostaining demonstrated that neuron specific enolase, GFAP and myelin basic protein (MBP) were present in HADFIL cells following the induction of differentiation, although one of the HADFIL cell populations showed a lower expression of GFAP and MBP. These results indicate that HADFIL cell populations have the potential to differentiate into neural cells. Although further studies are necessary to determine whether such in vitro-differentiated cells can function in vivo as neural cells, these amniotic cell populations might be of value in therapeutic applications that require human neural cells.

Impact, mechanisms, and novel chemotherapy strategies for overcoming resistance to anthracyclines and taxanes in metastatic breast cancer

Despite advances in treatment of patients with metastatic breast cancer (MBC), prognosis remains poor and median survival is 2-3 years. Resistance to antineoplastics mediated by many factors, potentially including overexpression of drug efflux proteins or altered beta-tubulin isotype expression limits the effectiveness of MBC chemotherapy. Capecitabine, approved for anthracycline- and taxane-resistant MBC, has produced modest responses, highlighting the need for more effective treatments for MBC resistant to multiple chemotherapeutic agents. Agents with potential to reverse drug resistance have not proven effective. Albumin-bound paclitaxel is a formulation that may enhance delivery to tumor tissues. Conversely, ixabepilone, an epothilone analog, has been reported to have lower susceptibility to at least some mechanisms of tumor resistance and clinical activity in resistant/refractory MBC. The topoisomerase-I inhibitor irinotecan also has low cross-resistance with other antineoplastics, and has shown some activity against refractory MBC. Development of new agents and identification of genetic biomarkers in translational studies promise to improve management of patients with resistant/refractory breast cancer.

Localization of βv tubulin in the cochlea and cultured cells with a novel monoclonal antibody

Tubulin, the dimeric structural protein of microtubules, is a heterodimer of alpha and beta subunits; both alpha and beta exist as numerous isotypes encoded by different genes. In vertebrates the sequence differences among the beta(I), beta(II), beta(III), beta(IV) and beta(V) isotypes are highly conserved in evolution, implying that the isotypes may have functional significance. Isotype-specific monoclonal antibodies have been useful in determining the cellular and sub-cellular distributions and possible functions of the beta(I), beta(II), beta(III), and beta(IV) isotypes; however, little is known about the beta(V) isotype. We here report the creation and purification of a monoclonal antibody (SHM.12G11) specific for beta(V). The antibody was designed to be specific for the C-terminal sequence EEEINE, which is unique to rodent and chicken beta(V). The antibody was found to bind specifically to the C-terminal peptide EEEINE, and does not cross-react with the carboxy-termini of either alpha-tubulin or the other beta-tubulin isotypes. However, the antibody also binds to the peptide EEEVNE, but not to the peptide EEEIDG, corresponding respectively to the C-terminal peptides of bovine and human beta(V). Immunofluorescence analysis indicates that beta(V) is found in microtubules of both the interphase network and the mitotic spindle. In gerbils, beta(V) also occurs in the cochlea where it is found largely in the specialized cells that are unique in containing bundled microtubules with 15 protofilaments.