Tubulin Loss Research Articles

Evaluation of tubulin β-3 and 5 hydroxy-methyl cytosine as diagnostic and prognostic markers in malignant melanoma

Tubulin β-3 staining pattern and staining intensity of 5-hydroxymethyl cytosine (5-hmC) are potential diagnostic and prognostic markers in melanocytic lesions that need further evaluation. Melanocytic nevi and primary cutaneous melanomas were immunohistochemically stained for tubulin-β-3 and 5-hmC. Immunoreactivity and staining patterns were correlated with Breslow-thickness, clinical and pathological characteristics, and progression-free survival. Melanocytes showed positive tubulin β-3 staining. However, in most nevi, tubulin β-3 staining appeared as a gradient with intense cytoplasmic staining in cells of the superficial part of the lesion that faded to weak staining in the deep dermal part, while no gradient was found in deep penetrating nevi and melanomas. In 53 % of the melanomas, areas with loss of tubulin β-3 staining were found. 5-hmC staining intensity was significantly higher in melanocytic nevi compared to melanomas. Breslow thickness in combination with low 5-hmC score and loss of tubulin-β-3 staining was predictive for poor prognosis. As single markers, tubulin-β-3 and 5-hmC can be useful to distinguish between melanocytic nevi and melanoma, but staining variability limits the use of 5-hmC. In melanomas measuring >1.5 mm, combination of low 5-hmC score and loss of tubulin-β-3 staining may have prognostic value.

151 TRPA1/V3-Dependent Regulation of Airway Epithelial Cell Damage and Repair Triggered by wood/Biomass Smoke Particulate

Abstract Wood/biomass smoke particulate material (WBSPM) is a pervasive environmental pollutant. Acute high-dose inhalation exposure can cause airway epithelial cell (AEC) damage and respiratory dysfunction. We hypothesized that AEC damage is limited by compensatory signaling to allow epithelial repair. RNA sequencing, proteomics, targeted interventions, and cultured human AECs treated with WBSPM and Transient Receptor Potential Ankyrin-1 (TRPA1) agonists revealed TRPA1-driven endoplasmic reticulum stress (ERS) and oxidative stress (OS) as contributory pathways to cytotoxicity. Alternatively, TRP vanilloid-3 (TRPV3) counteracted these events despite also being activated by WBSPM. Using the TRPA1 agonist AITC (and others) as a surrogate for WBSPM, morphological distortion indicative of AEC damage was observed 2-4h post-treatment; at >6h cells recovered. Strikingly, at 2h, the abundance of only 6 proteins changed including loss of fibronectin and tubulin and increased heme oxygenase 1. At 6h, 36 proteins changed, including ER chaperones and transcriptional/cell cycle regulatory proteins, OS-response, and structural/matrix proteins. Analysis of the phosphoproteome revealed changes in 2672 and 1886 unique phosphosites at 2 and 6h, attributable to PKC, p38 MAPK, EGFR, Akt and other kinase activities based on “phosphosite-GSEA” analysis. A kinase inhibitor screen linked PKC, p38 MAPK, GSK3b and ERK1/2 with cytotoxicity, while EGFR, MEK1/2, JNK1/2/3 and Akt were protective. Considering that TRPV3 expression is regulated by EGFR following WBSPM injury and TRPV3 enhanced adhesion and slowed proliferation via attenuated EGFR signaling, we propose a mechanism whereby the balance of TRPA1, TRPV3, and calcium/growth factor receptor-dependent kinase signaling coordinates WBSPM injury, compensation and repair.

Curcuma longa L. Prevents the Loss of \u03b2-Tubulin in the Brain and Maintains Healthy Aging in Drosophila melanogaster

Loss of tubulin is associated with neurodegeneration and brain aging. Turmeric (Curcuma longa L.) has frequently been employed as a spice in curry and traditional medications in the Indian subcontinent to attain longevity and better cognitive performance. We aimed to evaluate the unelucidated mechanism of how turmeric protects the brain to be an anti-aging agent. D. melanogaster was cultured on a regular diet and turmeric-supplemented diet. β-tubulin level and physiological traits including survivability, locomotor activity, fertility, tolerance to oxidative stress, and eye health were analyzed. Turmeric showed a hormetic effect, and 0.5% turmeric was the optimal dose in preventing aging. β-tubulin protein level was decreased in the brain of D. melanogaster upon aging, while a 0.5% turmeric-supplemented diet predominantly prevented this aging-induced loss of β-tubulin and degeneration of physiological traits as well as improved β-tubulin synthesis in the brain of D. melanogaster early to mid-age. The higher concentration (≥ 1%) of turmeric-supplemented diet decreased the β-tubulin level and degenerated many of the physiological traits of D. melanogaster. The turmeric concentration-dependent increase and decrease of β-tubulin level were consistent with the increment and decrement data obtained from the evaluated physiological traits. This correlation demonstrated that turmeric targets β-tubulin and has both beneficial and detrimental effects that depend on the concentration of turmeric. The findings of this study concluded that an optimal dosage of turmeric could maintain a healthy neuron and thus healthy aging, by preventing the loss and increasing the level of β-tubulin in the brain.

Docosahexaenoic acid protection in a rotenone induced Parkinson's model: Prevention of tubulin and synaptophysin loss, but no association with mitochondrial function

Rotenone, a classic mitochondrial complex I inhibitor, leads to dopaminergic neuronal death resulting in a Parkinson's-like-disease. Docosahexaenoic acid (DHA) has shown neuroprotective effects in other experimental models of Parkinson's disease, but its effect on the rotenone-induced parkinsonism is still unknown. We tested whether DHA in vivo exerts a neuroprotective effect on rotenone-induced parkinsonism and explored the mechanisms involved, including mitochondrial function and ultrastructure as well as the expression of tubulin and synaptophysin. We pretreated eighty male Wistar rats with DHA (35 mg/kg/day) for seven days and then administered rotenone for eight days. We then measured rearing behavior, number of dopaminergic neurons, tyrosine hydroxylase content, tubulin and synaptophysin expression, mitochondrial complex I, respiratory control ratio, mitochondrial transmembrane potential, ATP production activity and mitochondrial ultrastructure. We found that in vivo DHA supply exerted a neuroprotective effect, evidenced by decreased dopaminergic neuron cell death. Although we detected rotenone induced mitochondrial ultrastructure alterations, these were not associated with mitochondrial dysfunction. Rotenone had no effect on mitochondrial complex I, respiratory control ratio, mitochondrial transmembrane potential or ATP production activity. DHA also prevented a rotenone-induced decrease in tubulin and synaptophysin expression. Our results support the neuroprotective effect of DHA on rotenone-induced parkinsonism, and a possible effect on early stage Parkinson's disease. This protective effect is not associated with mitochondrial function improvement, but rather with preventing loss of tubulin and synaptophysin, proteins relevant to synaptic transmission.

Imbalanced Expression of Tau and Tubulin Induces Neuronal Dysfunction in C. elegans Models of Tauopathy.

Tauopathy is a type of dementia defined by the accumulation of filamentous tau inclusions in neural cells. Most types of dementia in the elderly, including Alzheimer’s disease, are tauopathies. Although it is believed that tau protein abnormalities and/or the loss of its functions results in neurodegeneration and dementia, the mechanism of tauopathy remains obscure. Loss of microtubules and/or tubulin is a known consequence of tau accumulating in neurons in Alzheimer’s disease. In other words, there is an excess level of tau relative to tubulin in tauopathy neurons. To test whether this imbalance of tau and tubulin expression results in the neurotoxicity of tau, we developed several transgenic C. elegans lines that express human tau at various levels in pan-neurons. These worms showed behavioral abnormalities in a tau expression-dependent manner. The knockdown of a tubulin-specific chaperon, or a subset of tubulin, led to enhanced tau toxicity even in low-expressing tau-transgenic worms that showed no abnormal behaviors. In addition, the suppression of tau expression in tubulin knockdown worms rescued neuronal dysfunction. Thus, not only the overexpression of tau but also a reduction in tubulin can trigger the neurotoxicity of tau. Tau expressed in worms was also highly phosphorylated and largely bound to tubulin dimers rather than microtubules. Relative amount of tubulin-unbound tau was increased in high-expressing tau-transgenic worms showing tau toxicity. We further demonstrated that tau aggregation was inhibited by co-incubation of purified tubulin in vitro, meaning sufficient amounts of tubulin can protect against the formation of tau inclusions. These results suggest that the expression ratio of tau to tubulin may be a determinant of the tauopathy cascade.

Abstract 036: Mitoprotection Preserves the Cytoskeleton and Alleviates Myocardial Injury in Early Swine Metabolic Syndrome

Background: The mechanisms responsible for cardiac injury in the early stage of metabolic syndrome (MetS) remain unknown. Mitochondria are intimately associated with myofibrils, where the cytoskeleton functions as a linkage coordinator. We hypothesized that early MetS is characterized by cytoskeletal-mitochondrial disorganization, and that mitoprotection with mitochondria-targeted peptides (MTP) would preserve cytoskeletal-mitochondrial structure and attenuate myocardial injury in early swine MetS. Methods: Pigs were studied after 16wks of diet-induced MetS, MetS treated for the last 4wks with the MTP Elamipretide (0.1mg/kg SC q.d), and Lean controls (n=6 each). Cardiac function (multidetector CT) was assessed in-vivo, and mitochondrial structure (electron microscopy), the mitochondrial inner membrane phospholipid cardiolipin (mass spectrometry), cytoskeletal proteins, oxidative stress, and apoptosis ex-vivo. Results: MetS pigs developed hypertension and insulin resistance, yet cardiac function was preserved. MetS induced loss of desmin and tubulin that was paralleled by mitochondrial disorganization, decreased 18:2 cardiolipin, and increased oxidative stress and apoptosis. MTP slightly improved cardiolipin species profile, restored mitochondrial organization, and upregulated cytoskeletal proteins, attenuating apoptosis and oxidative stress. Conclusion: Early MetS leads to disorganization of the cardiomyocyte cytoskeletal- mitochondrial architecture and cardiac injury. MTP may provide a novel therapeutic potential for improving cardiac injury in early MetS, and potentially preventing future deterioration in cardiac function.

Array tomography for the detection of non-dilated, injured axons in traumatic brain injury

BackgroundAxonal injury is a key feature of several types of brain trauma and neurological disease. However, in mice and humans, many axons are less than 500nm in diameter which is at or below the resolution of most conventional light microscopic imaging methods. In moderate to severe forms of axon injury, damaged axons become dilated and therefore readily detectible by light microscopy. However, in more subtle forms of injury, the damaged axons may remain undilated and therefore difficult to detect. New methodHere we present a method for adapting array tomography for the identification and quantification of injured axons. In this technique, ultrathin (∼70nm) plastic sections of tissue are prepared, labeled with axon injury-relevant antibodies and imaged using conventional epifluorescence. ResultsTo demonstrate the use of array-tomography-based methods, we determined that mice that received two closed-skull concussive traumatic brain injury impacts had significantly increased numbers of non-dilated axons that were immunoreactive for non-phosphorylated neurofilament (SMI-32; a marker of axonal injury), compared to sham mice (1682±628 versus 339±52 per mm2, p=0.004, one-tailed Mann–Whitney U test). Tubulin loss was not evident (p=0.2063, one-tailed Mann–Whitney U test). Furthermore, mice that were subjected to more severe injury had a loss of tubulin in addition to both dilated and non-dilated SMI-32 immunoreactive axons indicating that this technique is suitable for the analysis of various injury conditions. Comparison with existing methodWith array tomography we could detect similar overall numbers of axons as electron microscopy, but accurate diameter measurements were limited to those with diameters >200nm. Importantly, array tomography had greater sensitivity for detecting small non-dilated injured axons compared with conventional immunohistochemistry. ConclusionImaging of individual axons and quantification of subtle axonal injury is possible using this array tomography method. This method may be most useful for the assessment of concussive injuries and other pathologies in which injured axons are not typically dilated. The ability to process moderately large volumes of tissue, label multiple proteins of interest, and automate analysis support array tomography as a useful alternative to electron microscopy.

Modeling the effects of drug binding on the dynamic instability of microtubules

We propose a stochastic model that accounts for the growth, catastrophe and rescue processes of steady-state microtubules assembled from MAP-free tubulin in the possible presence of a microtubule-associated drug. As an example of the latter, we both experimentally and theoretically study the perturbation of microtubule dynamic instability by S-methyl-D-DM1, a synthetic derivative of the microtubule-targeted agent maytansine and a potential anticancer agent. Our model predicts that among the drugs that act locally at the microtubule tip, primary inhibition of the loss of GDP tubulin results in stronger damping of microtubule dynamics than inhibition of GTP tubulin addition. On the other hand, drugs whose action occurs in the interior of the microtubule need to be present in much higher concentrations to have visible effects.

Patronin Regulates the Microtubule Network by Protecting Microtubule Minus Ends

Tubulin assembles into microtubule polymers that have distinct plus and minus ends. Most microtubule plus ends in living cells are dynamic; the transitions between growth and shrinkage are regulated by assembly-promoting and destabilizing proteins. In contrast, minus ends are generally not dynamic, suggesting their stabilization by some unknown protein. Here, we have identified Patronin (also known as ssp4) as a protein that stabilizes microtubule minus ends in Drosophila S2 cells. In the absence of Patronin, minus ends lose subunits through the actions of the Kinesin-13 microtubule depolymerase, leading to a sparse interphase microtubule array and short, disorganized mitotic spindles. In vitro, the selective binding of purified Patronin to microtubule minus ends is sufficient to protect them against Kinesin-13-induced depolymerization. We propose that Patronin caps and stabilizes microtubule minus ends, an activity that serves a critical role in the organization of the microtubule cytoskeleton.

Abstract 4421: The ability of the microtubule-targeting agent T0070907 to induce tubulin degradation in colorectal cancer cell lines is affected by the microtubule-destabilizing but not the tubulin sequestering functions of stathmin

Abstract Background: The compound T0070907 is a microtubule-targeting agent (MTA) that, unlike standard MTAs, induces proteasomal degradation of tubulin. Strikingly, tubulin loss occurs preferentially in tumor cells. One potential candidate for a tumor-specific function that affects the ability of T0070907 to induce tubulin loss is the microtubule-associated protein stathmin. Stathmin normally destabilizes microtubules and also sequesters tubulin in complexes unavailable for degradation. We therefore examined whether T0070907-induced stathmin inactivation is required for tubulin loss. Methods: 50μM T0070907 was added to the colorectal cancer cell lines HT-29, HCT-15, and SW620, and microtubule polymerization, tubulin loss, and stathmin phosphorylation measured at multiple time points by Western blot. When applicable, microtubules were depolymerized with 0.1 μM vinblastine or nocodazole or polymerized with 0.1 μM paclitaxel before addition of T0070907. Microtubule polymerization was quantified by cell lysis in microtubule stabilizing buffer followed by centrifugation to separate the insoluble microtubules from the soluble tubulin heterodimers. Stathmin levels were reduced using chemically synthesized stathmin-specific siRNAs; responsiveness to T0070907 was assessed after 72h knockdown. Results: T0070907 induced tubulin loss starting 8h after compound addition. Depolymerizing the microtubules eliminated the lag time before tubulin loss, while polymerizing the microtubules with paclitaxel prevented loss. T0070907 alone or in the presence of 25μM of the proteasome inhibitor MG132 increased the amount of tubulin found in the soluble, degradable fraction. T0070907 also increased phosphorylation of the stathmin regulatory ser16 site that normally inactivates both microtubule destabilizing and sequestering functions. Stathmin knockdown slowed the kinetics of T0070907-induced tubulin loss, as was expected given that stathmin normally destabilizes microtubules. However, stathmin knockdown did not increase the rate of T0070907-induced tubulin loss when microtubules were fully depolymerized with vinblastine, as would be expected if inactivation of the sequestering function were critical. Conclusion: T0070907-induced degradation requires microtubule depolymerization of the microtubules. T0070907 increases depolymerization, and also inactivates stathmin. However, this inactivation does not contribute to the ability of T0070907 to degrade tubulin, as inactivation of the stathmin sequestering function does not seem to be critical to T0070907's ability to degrade tubulin, and inactivation of the microtubule destabilizing function antagonizes tubulin degradation. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. 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 4421.

Highly Variable Microtubule Assembly Dynamics Reflect Near-Kilohertz Kinetics: Evidence Against Traditional Linear Growth Theory

Microtubules are intracellular polymers that dynamically grow and shorten at their ends via the stochastic addition and loss of alpha-beta-tubulin heterodimers. The kinetics of tubulin assembly are central to the regulation of microtubule dynamics by microtubule-associated proteins and therapeutic drugs. Previously, rates of tubulin subunit exchange at the ends of growing microtubules have been estimated using a linear growth theory that assumes tubulin dissociation occurs at a constant rate regardless of the free tubulin concentration. However, by measuring the variance of microtubule assembly at the nanometer scale, we find that stochastic tubulin dynamics are an order of magnitude faster than previously estimated. This discrepancy is explained by molecular-level simulations showing that the tubulin dissociation rate during microtubule growth is not constant, but rather should increase with increasing tubulin concentration. This indirect effect is due to a concentration-dependent bias in simulated microtubule tip structure, as has been experimentally observed. Our analyses indicate that the published tubulin subunit addition and loss rates at growing microtubule ends in vitro have been consistently underestimated in the literature: the variance in the assembly rate in vitro is too high to be consistent with the previous low kinetic rate estimates, and we conclude that both tubulin addition and tubulin loss events occur on the millisecond time scale, far faster than the previously believed 10-1000 millisecond scale. More generally, we demonstrate that the fixed off rate originally used in the linear growth theory of Oosawa and assumed in most subsequent models, fails to describe the behavior of self-assembled polymers having both lateral and longitudinal bonding interactions between subunits.

The microtubule-targeting agent T0070907 induces proteasomal degradation of tubulin

Current microtubule-targeting agents interfere with the regulated assembly of microtubules from α/β tubulin heterodimers but do not markedly alter tubulin levels. Previously, we showed that the compound T0070907 interferes with microtubule function by reversibly decreasing α and β tubulin protein levels by more than 50% in multiple CRC cell lines. Since tubulin levels are generally relatively stable, and cells lack regulatory networks to respond to decreased tubulin levels by increasing synthesis, our result suggested the possibility of cancer therapies that act directly on tubulin homeostasis. The aim of this study was to determine whether T0070907 caused tubulin loss by increasing the degradation rate, and determine the proteases responsible for any increased degradation. T0070907 increased tubulin degradation rates in HT-29 cells. The proteasomal inhibitors MG132, epoxomicin, lactacystin, and ALLN suppressed T0070907-mediated tubulin loss, although epoxomicin and lactacystin were less effective than MG132, even at concentrations that completely inhibited TNFα-induced IκBα degradation. Inhibitors of lysosomal, aggresomal, and calpain-mediated degradation, as well as the caspase inhibitor zVAD-fmk had no effect on tubulin loss, and the cathepsin and calpain inhibitor E64d was unable to increase epoxomicin’s ability to suppress tubulin loss. We conclude that T0070907-induced tubulin degradation proceeds through a proteasome-dependent pathway.

Inhibition of Tau Polymerization with a Cyanine Dye in Two Distinct Model Systems

In a host of neurodegenerative diseases Tau, a microtubule-associated protein, aggregates into insoluble lesions within neurons. Previous studies have utilized cyanine dyes as Tau aggregation inhibitors in vitro. Herein we utilize cyanine dye 3,3'-diethyl-9-methyl-thiacarbocyanine iodide (C11) to modulate Tau polymerization in two model systems, an organotypic slice culture model derived from Tau transgenic mice and a split green fluorescent protein complementation assay in Tau-expressing cells. In slice cultures, submicromolar concentrations (0.001 microm) of C11 produced a significant reduction of aggregated Tau and a corresponding increase in unpolymerized Tau. In contrast, treatment with a 1 microm dose promoted aggregation of Tau. These results were recapitulated in the complementation assay where administration of 1 microm C11 produced a significant increase in polymerized Tau relative to control, whereas treatment of cells with 0.01 microm C11 resulted in a marked reduction of aggregated Tau. In the organotypic slice cultures, modulation of Tau aggregation was independent of changes in phosphorylation at disease and microtubule binding relevant epitopes for both dosing regimes. Furthermore, treatment with 0.001 microm C11 resulted in a decrease in both total filament mass and number. There was no evidence of apoptosis or loss of synaptic integrity at either dose, however, whereas submicromolar concentrations of C11 did not interfere with microtubule binding, higher doses resulted in a decrease in the levels of microtubule-bound Tau. Overall, a cyanine dye can dissociate aggregated Tau in an ex vivo model of tauopathy with little toxicity and exploration of the use of these type of dyes as therapeutic agents is warranted.

Regulation of Microtubule Dynamic Instability in Vitro by Differentially Phosphorylated Stathmin

Stathmin is an important regulator of microtubule polymerization and dynamics. When unphosphorylated it destabilizes microtubules in two ways, by reducing the microtubule polymer mass through sequestration of soluble tubulin into an assembly-incompetent T2S complex (two alpha:beta tubulin dimers per molecule of stathmin), and by increasing the switching frequency (catastrophe frequency) from growth to shortening at plus and minus ends by binding directly to the microtubules. Phosphorylation of stathmin on one or more of its four serine residues (Ser(16), Ser(25), Ser(38), and Ser(63)) reduces its microtubule-destabilizing activity. However, the effects of phosphorylation of the individual serine residues of stathmin on microtubule dynamic instability have not been investigated systematically. Here we analyzed the effects of stathmin singly phosphorylated at Ser(16) or Ser(63), and doubly phosphorylated at Ser(25) and Ser(38), on its ability to modulate microtubule dynamic instability at steady-state in vitro. Phosphorylation at either Ser(16) or Ser(63) strongly reduced or abolished the ability of stathmin to bind to and sequester soluble tubulin and its ability to act as a catastrophe factor by directly binding to the microtubules. In contrast, double phosphorylation of Ser(25) and Ser(38) did not affect the binding of stathmin to tubulin or microtubules or its catastrophe-promoting activity. Our results indicate that the effects of stathmin on dynamic instability are strongly but differently attenuated by phosphorylation at Ser(16) and Ser(63) and support the hypothesis that selective targeting by Ser(16)-specific or Ser(63)-specific kinases provides complimentary mechanisms for regulating microtubule function.

695 Sulforaphane Sensitizes Colorectal Cancer Cells to Oxaliplatin Induced Cell Growth Inhibition: Key Role of p38 MAPK

Background: Microtubule-targeting agents (MTAs) are cancer drugs that act primarily by altering microtubule dynamics and interfering with mitosis. While MTAs are in widespread clinical use, innate and acquired resistance is an ongoing problem. Resistance is thought to stem primarily from drug efflux and expression of alternate tubulin isoforms with MTAresistant microtubule dynamics. We found that the compound T0070907 reduced tumor growth in a murine xenograft model of colon cancer. In human colorectal cancer (CRC) cell lines, T0070907 caused near-total loss of the α and β tubulin proteins, leading to collapse of visible microtubule filaments and eventual cell death. Dramatic tubulin loss is a novel phenotype not reported for any of the standard MTAs. This observation suggests the possible development of MTAs that work by a mechanism less susceptible to escape via expression of alternate tubulin isoforms. As a first step toward determining the molecular mechanisms of tubulin loss, we determined whether T0070907 affects degradation and activity of the microtubule associated proteins that control microtubule polymerization. Methods: The effects of T0070907 on tubulin levels and phosphorylation of the microtubuleassociated protein stathmin/Op18 and its upstream kinase MARK kinase were examined using Western blot in the CRC cell lines HT-29, SW620, and DLD-1. Tubulin protein halflives were measured after addition of the protein synthesis inhibitor cycloheximide. Results: T0070907 caused loss of α and β, but not γ, δ, or e tubulin. The magnitude of the tubulin reduction was much greater than that found when microtubule destabilization leads to tubulin synthesis inhibition. Tubulin half lives decreased from >12h to 6h, suggesting increased degradation. Consistent with this idea, treatments that sequestered tubulin in polymerized microtubules (paclitaxel), leaving the tubulin unavailable for degradation, reduced T0070907-induced tubulin loss. Conversely, the microtubule-destabilizing agents nocodazole and vinblastine increased the rate of tubulin loss. T0070907 did not strongly affect microtubule polymerization In Vitro, suggesting that it does not directly bind to either microtubules or tubulin. However, T0070907 led to increases in MARK kinase activation as well as phosphorylation and inactivation of its downstream target, the microtubulesequestering protein stathmin/Op18. Conclusion: T0070907 promotes tubulin degradation and stathmin inactivation. As knockdown of stathmin in Drosophila leads to a drastic reduction in tubulin levels, we propose that T0070907 reduces tubulin in part by targeting stathmin.

696 Resveratrol Sensitizes Colorectal Cancer Cells to Oxaliplatin-Induced Cell Death via Intracellular Polyamine Depletion

Background: Microtubule-targeting agents (MTAs) are cancer drugs that act primarily by altering microtubule dynamics and interfering with mitosis. While MTAs are in widespread clinical use, innate and acquired resistance is an ongoing problem. Resistance is thought to stem primarily from drug efflux and expression of alternate tubulin isoforms with MTAresistant microtubule dynamics. We found that the compound T0070907 reduced tumor growth in a murine xenograft model of colon cancer. In human colorectal cancer (CRC) cell lines, T0070907 caused near-total loss of the α and β tubulin proteins, leading to collapse of visible microtubule filaments and eventual cell death. Dramatic tubulin loss is a novel phenotype not reported for any of the standard MTAs. This observation suggests the possible development of MTAs that work by a mechanism less susceptible to escape via expression of alternate tubulin isoforms. As a first step toward determining the molecular mechanisms of tubulin loss, we determined whether T0070907 affects degradation and activity of the microtubule associated proteins that control microtubule polymerization. Methods: The effects of T0070907 on tubulin levels and phosphorylation of the microtubuleassociated protein stathmin/Op18 and its upstream kinase MARK kinase were examined using Western blot in the CRC cell lines HT-29, SW620, and DLD-1. Tubulin protein halflives were measured after addition of the protein synthesis inhibitor cycloheximide. Results: T0070907 caused loss of α and β, but not γ, δ, or e tubulin. The magnitude of the tubulin reduction was much greater than that found when microtubule destabilization leads to tubulin synthesis inhibition. Tubulin half lives decreased from >12h to 6h, suggesting increased degradation. Consistent with this idea, treatments that sequestered tubulin in polymerized microtubules (paclitaxel), leaving the tubulin unavailable for degradation, reduced T0070907-induced tubulin loss. Conversely, the microtubule-destabilizing agents nocodazole and vinblastine increased the rate of tubulin loss. T0070907 did not strongly affect microtubule polymerization In Vitro, suggesting that it does not directly bind to either microtubules or tubulin. However, T0070907 led to increases in MARK kinase activation as well as phosphorylation and inactivation of its downstream target, the microtubulesequestering protein stathmin/Op18. Conclusion: T0070907 promotes tubulin degradation and stathmin inactivation. As knockdown of stathmin in Drosophila leads to a drastic reduction in tubulin levels, we propose that T0070907 reduces tubulin in part by targeting stathmin.

694 The Tubulin-Targeting Agent T0070907 Promotes Tubulin Degradation and Inactivates the Tubulin-Sequestering Protein Stathmin/Op18

Background: Microtubule-targeting agents (MTAs) are cancer drugs that act primarily by altering microtubule dynamics and interfering with mitosis. While MTAs are in widespread clinical use, innate and acquired resistance is an ongoing problem. Resistance is thought to stem primarily from drug efflux and expression of alternate tubulin isoforms with MTAresistant microtubule dynamics. We found that the compound T0070907 reduced tumor growth in a murine xenograft model of colon cancer. In human colorectal cancer (CRC) cell lines, T0070907 caused near-total loss of the α and β tubulin proteins, leading to collapse of visible microtubule filaments and eventual cell death. Dramatic tubulin loss is a novel phenotype not reported for any of the standard MTAs. This observation suggests the possible development of MTAs that work by a mechanism less susceptible to escape via expression of alternate tubulin isoforms. As a first step toward determining the molecular mechanisms of tubulin loss, we determined whether T0070907 affects degradation and activity of the microtubule associated proteins that control microtubule polymerization. Methods: The effects of T0070907 on tubulin levels and phosphorylation of the microtubuleassociated protein stathmin/Op18 and its upstream kinase MARK kinase were examined using Western blot in the CRC cell lines HT-29, SW620, and DLD-1. Tubulin protein halflives were measured after addition of the protein synthesis inhibitor cycloheximide. Results: T0070907 caused loss of α and β, but not γ, δ, or e tubulin. The magnitude of the tubulin reduction was much greater than that found when microtubule destabilization leads to tubulin synthesis inhibition. Tubulin half lives decreased from >12h to 6h, suggesting increased degradation. Consistent with this idea, treatments that sequestered tubulin in polymerized microtubules (paclitaxel), leaving the tubulin unavailable for degradation, reduced T0070907-induced tubulin loss. Conversely, the microtubule-destabilizing agents nocodazole and vinblastine increased the rate of tubulin loss. T0070907 did not strongly affect microtubule polymerization In Vitro, suggesting that it does not directly bind to either microtubules or tubulin. However, T0070907 led to increases in MARK kinase activation as well as phosphorylation and inactivation of its downstream target, the microtubulesequestering protein stathmin/Op18. Conclusion: T0070907 promotes tubulin degradation and stathmin inactivation. As knockdown of stathmin in Drosophila leads to a drastic reduction in tubulin levels, we propose that T0070907 reduces tubulin in part by targeting stathmin.

Juglone Inactivates Cysteine-rich Proteins Required for Progression through Mitosis

The parvulin peptidyl-prolyl isomerase Pin1 catalyzes cis-trans isomerization of p(S/T)-P bonds and might alter conformation and function of client proteins. Since the trans conformation of p(S/T)-P bonds is preferred by protein phosphatase 2A (PP2A), Pin1 may facilitate PP2A-mediated dephosphorylation. Juglone irreversibly inhibits parvulins and is often used to study the function of Pin1 in vivo. The drug prevents dephosphorylation of mitotic phosphoproteins, perhaps because they bind Pin1 and are dephosphorylated by PP2A. We show here however that juglone inhibited post-mitotic dephosphorylation and the exit of mitosis, independent of Pin1. This effect involved covalent modification of sulfhydryl groups in proteins essential for metaphase/anaphase transition. Particularly cytoplasmic proteins with a high cysteine content were vulnerable to the drug. Alkylation of sulfhydryl groups altered the conformation of such proteins, as evidenced by the disappearance of antibody epitopes on tubulin and the mitotic checkpoint component BubR1. The latter activates the anaphase-promoting complex/cyclosome, which degrades regulatory proteins, such as cyclin B1 and securins, and is required for mitotic exit. Indeed, juglone-treated cells failed to assemble a mitotic spindle, which correlated with perturbed microtubule dynamics, loss of immunodetectable tubulin, and formation of tubulin aggregates. Juglone also prevented degradation of cyclin B1, independently of the Mps1-controlled mitotic spindle checkpoint. Since juglone affected cell cycle progression at several levels, more specific drugs need to be developed for studies of Pin1 function in vivo.

PPARγ inhibitors reduce tubulin protein levels by a PPARγ, PPARδ and proteasome‐independent mechanism, resulting in cell cycle arrest, apoptosis and reduced metastasis of colorectal carcinoma cells

The nuclear transcription factor peroxisome proliferator-activated receptor-gamma (PPARgamma) has been identified as an important therapeutic target in murine models of colorectal cancer (CRC). To examine whether PPARgamma inhibition has therapeutic effects in late-stage CRC, the effects of PPARgamma inhibitors on CRC cell survival were examined in CRC cell lines and a murine CRC model. Low doses (0.1-1 microM) of PPARgamma inhibitors (T0070907, GW9662 and BADGE) did not affect cell survival, while higher doses (10-100 microM) of all 3 PPARgamma inhibitors caused caspase-dependent apoptosis in HT-29, Caco-2 and LoVo CRC cell lines. Apoptosis was preceded by altered cell morphology, and this alteration was not prevented by caspase inhibition. PPARgamma inhibitors also caused dual G and M cell cycle arrest, which was not required for apoptosis or for morphologic alterations. Furthermore, PPARgamma inhibitors triggered loss of the microtubule network. Notably, unlike other standard antimicrotubule agents, PPARgamma inhibitors caused microtubule loss by regulating tubulin post-transcriptionally rather than by altering microtubule polymerization or dynamics. Proteasome inhibition by epoxomicin was unable to prevent tubulin loss. siRNA-mediated reduction of PPARgamma and PPARdelta proteins did not replicate the effects of PPARgamma inhibitors or interfere with the inhibitors' effects on apoptosis, cell cycle or tubulin. PPARgamma inhibitors also reduced CRC cell migration and invasion in assays in vitro and reduced both the number and size of metastases in a HT-29/SCID xenograft metastatic model of CRC. These results suggest that PPARgamma inhibitors are a novel potential antimicrotubule therapy for CRC that acts by directly reducing microtubule precursors.

Screening for microtubule-disrupting antifungal agents by using a mitotic-arrest mutant of Aspergillus nidulans and novel action of phenylalanine derivatives accompanying tubulin loss.

The microtubule, which is one of the major targets of anthelmintics, anticancer drugs, and fungicides, is composed mainly of alpha- and beta-tubulins. We focused on a unique characteristic of an Aspergillus nidulans benA33 mutant to screen for microtubule-disrupting antifungal agents. This mutant, which has a beta-tubulin with a mutation of a single amino acid, undergoes mitotic arrest due to the formation of hyperstable microtubules at 37 degrees C. The heat sensitivity of the mutant is remedied by some antimicrotubule agents. We found that an agar plate assay with the mutant was able to distinguish three types of microtubule inhibitors. The growth recovery zones of the mutant were formed around paper disks containing microtubule inhibitors, including four benzimidazoles, ansamitocin P-3, griseofulvin, and rhizoxin, on the agar plate at 37 degrees C. Nocodazole, thiabendazole, and griseofulvin reversed the mitotic arrest of the mutant and promoted its hyphal growth. Ansamitocin P-3 and rhizoxin showed growth recovery zones around the growth-inhibitory zones. Benomyl and carbendazim also reversed mitotic arrest but produced weaker growth recovery than the aforementioned drugs. Other microtubule inhibitors, such as colchicine, Colcemid, paclitaxel, podophyllotoxin, TN-16, vinblastine, and vincristine, as well as some cytoskeletal inhibitors tested, did not show such activity. In our screening, we newly identified two mycotoxins, citrinin and patulin, two sesquiterpene dialdehydes, polygodial and warburganal, and four phenylalanine derivatives, arphamenine A, L-2,5-dihydrophenylalanine (DHPA), N-tosyl-L-phenylalanine chloromethylketone, and N-carbobenzoxy-L-phenylalanine chloromethyl ketone. In a wild-type strain of A. nidulans, DHPA caused selective losses of microtubules, as determined by fluorescence microscopy, and of both alpha- and beta-tubulins, as determined by Western blot analysis. This screening method involving the benA33 mutant of A. nidulans is useful, convenient, and highly selective. The phenylalanine derivatives tested are of a novel type of microtubule-disrupting antifungal agents, producing an accompanying loss of tubulins, and are different from well-known tubulin inhibitors affecting the assembly of tubulin dimers into microtubules.