Microtubule-depolymerizing Drug Research Articles

Abstract 510: Flow cytometry evaluation of cell cycle arrest in A549 human non-small cell lung cancer cells and tumors following treatment with cell cycle inhibitors: A multifaceted approach to targeting cancer

Abstract The contemporary landscape of cancer treatment has witnessed significant progress with FDA-approved targeted drugs. The FDA's Project Optimus has underscored the growing need for innovative pharmacodynamic endpoints to determine the optimal biological dose in clinical development. Flow cytometry, a potent analytical tool in clinical and preclinical labs, now plays a leading role in cancer research, enabling the evaluation of new drug effects on cell cycle regulation and consideration of rational drug combinations with approved drugs. At TD2, we strive to enhance the translatability of in vitro induced cell cycle arrest studies to in vivo biomarker proof of concept and efficacy. We employ a comprehensive approach, utilizing flow cytometry to assess cell cycle dynamics in cultured cells and in vivo tumor models after treatment with approved and novel drugs. This abstract focuses on the translational relevance of combined in vitro and in vivo testing of well-known drugs that disrupt the cell cycle, laying the foundation for optimized treatment strategies and potentially personalized cancer therapies. Human A549 non-small cell lung carcinoma cells were treated with either thymidine (2.0mM) or the microtubule-depolymerizing drug nocodazole (100ng/ml) for 18 hours, then examined by flow cytometry. Thymidine induced a greater than 85% S-phase arrest, while nocodazole induced a greater than 88% G2/M-phase arrest. A549 cells (5 × 106 cells/mouse) were subcutaneously implanted in SCID mice, followed by treatment with an FDA-approved cell cycle inhibitor, Palbociclib, or cisplatin chemotherapy. Palbociclib, a CDK4/6 inhibitor, and low-dose cisplatin treatment resulted in an increased accumulation of cells in the G0/G1 phase, greater than 80% for each. Both treatments exhibited promising anti-cancer activity, with tumor growth inhibition (TGI) of 69% and 87%, respectively. Our findings highlight flow cytometry's multifaceted utility in assessing cell cycle arrest, providing a comprehensive view of agent effects on cancer cells in culture and within the complex in vivo tumor microenvironment. Determining the impact of any new drug on specific cell cycle checkpoints could allow for a more strategic plan for combination therapy. Integrating flow cytometry could enable early biomarker monitoring of tumor cell cycle changes, offering insights into the therapeutic potential of new agents alone or in combination. This multidimensional evaluation contributes to understanding cancer biology and has the potential to guide the development of novel therapeutic strategies, optimize treatment regimens to identify optimal biological doses, and improve patient outcomes in the era of precision medicine. Citation Format: Erin E. Trachet, Emily Cluff, Ana Lemos de Matos, Damaris Diaz, Chelsea Holmquist, Paul Gonzales. Flow cytometry evaluation of cell cycle arrest in A549 human non-small cell lung cancer cells and tumors following treatment with cell cycle inhibitors: A multifaceted approach to targeting cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 510.

Microtubule disruption reduces metastasis more effectively than primary tumor growth

Clinical cancer imaging focuses on tumor growth rather than metastatic phenotypes. The microtubule-depolymerizing drug, Vinorelbine, reduced the metastatic phenotypes of microtentacles, reattachment and tumor cell clustering more than tumor cell viability. Treating mice with Vinorelbine for only 24 h had no significant effect on primary tumor survival, but median metastatic tumor survival was extended from 8 to 30 weeks. Microtentacle inhibition by Vinorelbine was also detectable within 1 h, using tumor cells isolated from blood samples. As few as 11 tumor cells were sufficient to yield 90% power to detect this 1 h Vinorelbine drug response, demonstrating feasibility with the small number of tumor cells available from patient biopsies. This study establishes a proof-of-concept that targeted microtubule disruption can selectively inhibit metastasis and reveals that existing FDA-approved therapies could have anti-metastatic actions that are currently overlooked when focusing exclusively on tumor growth.

Epistasis, aneuploidy, and functional mutations underlie evolution of resistance to induced microtubule depolymerization.

Cells with blocked microtubule polymerization are delayed in mitosis, but eventually manage to proliferate despite substantial chromosome missegregation. While several studies have analyzed the first cell division after microtubule depolymerization, we have asked how cells cope long-term with microtubule impairment. We allowed 24 clonal populations of yeast cells with beta-tubulin mutations preventing proper microtubule polymerization, to evolve for ˜150 generations. At the end of the laboratory evolution experiment, cells had regained the ability to form microtubules and were less sensitive to microtubule-depolymerizing drugs. Whole-genome sequencing identified recurrently mutated genes, in particular for tubulins and kinesins, as well as pervasive duplication of chromosome VIII. Recreating these mutations and chromosome VIII disomy prior to evolution confirmed that they allow cells to compensate for the original mutation in beta-tubulin. Most of the identified mutations did not abolish function, but rather restored microtubule functionality. Analysis of the temporal order of resistance development in independent populations repeatedly revealed the same series of events: disomy of chromosome VIII followed by a single additional adaptive mutation in either tubulins or kinesins. Since tubulins are highly conserved among eukaryotes, our results have implications for understanding resistance to microtubule-targeting drugs widely used in cancer therapy.

Dynein- and kinesin- mediated intracellular transport on microtubules facilitates RABV infection

Rabies, caused by rabies virus (RABV), is one of the most important neurotropic zoonoses and poses a severe threat to human and animal health. Exploration of its mechanism of neural transmission is meaningful but still insufficient. Here, we described the effects of microtubule-depolymerizing drugs and inhibitors of microtubule motor proteins on RABV infection. Colchicine, a microtubule-depolymerizing drug, significantly impeded RABV production in N2a cells. Overexpression of CC1 or p50 attenuated viral infection through the functional disruption of cytoplasmic dynein, which was consistent with the inhibitory effect of Na3VO4, a dynein activity inhibitor. Moreover, transfection with Flag-KHCct impaired RABV infection, as cytoplasmic kinesin-based motility was blocked. These results demonstrated that RABV can infect N2a cells in a manner that depends on microtubule integrity as well as dynein and kinesin function.

External Mechanical Cues Reveal a Katanin-Independent Mechanism behind Auxin-Mediated Tissue Bending in Plants.

Tissue folding is a central building block of plant and animal morphogenesis. In dicotyledonous plants, hypocotyl folds to form hooks after seedling germination that protects their aerial stem cell niche during emergence from soil. Auxin response factors and auxin transport are reported to play a key role in this process. Here, we show that the microtubule-severing enzyme katanin contributes to hook formation. However, by exposing hypocotyls to external mechanical cues mimicking the natural soil environment, we reveal that auxin response factors ARF7/ARF19, auxin influx carriers, and katanin are dispensable for apical hook formation, indicating that these factors primarily play the role of catalyzers of tissue bending in the absence of external mechanical cues. Instead, our results reveal the key roles of the non-canonical TMK-mediated auxin pathway, PIN efflux carriers, and cellulose microfibrils as components of the core pathway behind hook formation in the presence or absence of external mechanical cues.

FGF401 and vinorelbine synergistically mediate antitumor activity and vascular normalization in FGF19-dependent hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is a lethal cancer with limited therapeutic options, and standard therapy with sorafenib provides only modest survival benefits. Fibroblast growth factor 19 (FGF19) has been proposed as a driver oncogene, and targeting its receptor, FGFR-4, may provide a better alternative to standard therapy for patients with FGF19-driven tumors. Sixty-three HCC patient-derived xenograft (PDX) models were screened for FGF19 expression. Mice bearing high and low FGF19-expressing tumors were treated with FGF401 and/or vinorelbine, and the antitumor activity of both agents was assessed individually and in combination. Tumor vasculature and intratumoral hypoxia were also examined. High FGF19 expression was detected in 14.3% (9 of 63) of the HCC models tested and may represent a good target for HCC treatment. FGF401 potently inhibited the growth of high FGF19-expressing HCC models regardless of FGF19 gene amplification. Furthermore, FGF401 inhibited the FGF19/FGFR-4 signaling pathway, cell proliferation, and hypoxia, induced apoptosis and blood vessel normalization and prolonged the overall survival (OS) of mice bearing high FGF19 tumors. FGF401 synergistically acted with the microtubule-depolymerizing drug vinorelbine to further suppress tumor growth, promote apoptosis, and prolong the OS of mice bearing high FGF19 tumors, with no evidence of increased toxicity. Our study suggests that a subset of patients with high FGF19-expressing HCC tumors could benefit from FGF401 or FGF401/vinorelbine treatment. A high level of FGF19 in a tumor may serve as a potential biomarker for patient selection.

Synchronization of human retinal pigment epithelial-1 cells in mitosis.

ABSTRACTHuman retinal pigment epithelial-1 (RPE-1) cells are increasingly being used as a model to study mitosis because they represent a non-transformed alternative to cancer cell lines, such as HeLa cervical adenocarcinoma cells. However, the lack of an efficient method to synchronize RPE-1 cells in mitosis precludes their application for large-scale biochemical and proteomics assays. Here, we report a protocol to synchronize RPE-1 cells based on sequential treatments with the Cdk4 and Cdk6 inhibitor PD 0332991 (palbociclib) and the microtubule-depolymerizing drug nocodazole. With this method, the vast majority (80–90%) of RPE-1 cells arrested at prometaphase and exited mitosis synchronously after release from nocodazole. Moreover, the cells fully recovered and re-entered the cell cycle after the palbociclib–nocodazole block. Finally, we show that this protocol could be successfully employed for the characterization of the protein–protein interaction network of the kinetochore protein Ndc80 by immunoprecipitation coupled with mass spectrometry. This synchronization method significantly expands the versatility and applicability of RPE-1 cells to the study of cell division and might be applied to other cell lines that do not respond to treatments with DNA synthesis inhibitors.

Mal3 is a multi-copy suppressor of the sensitivity to microtubule-depolymerizing drugs and chromosome mis-segregation in a fission yeast pka1 mutant.

The cAMP-dependent protein kinase Pka1 is known as a regulator of glycogenesis, transition into meiosis, chronological aging, and stress responses in the fission yeast, Schizosaccharomyces pombe. We demonstrated here that Pka1 is responsible for normal growth in the presence of the microtubule-destabilization drug TBZ and proper chromosome segregation. The deletion of the pka1 gene resulted in the TBZ-sensitive phenotype and chromosome mis-segregation. We isolated the mal3 gene as a multi-copy suppressor of the TBZ-sensitive phenotype in the pka1Δ strains. Overexpression of the CH domain (1–143) or the high-affinity microtubule binding mutant (1–143 Q89R) of Mal3 rescued the TBZ-sensitive phenotype in the pka1Δ and mal3Δ strains, while the EB1 domain (135–308) and the mutants defective in microtubule binding (1–143 Q89E) failed to do so in the same strains. Chromosome mis-segregation caused by TBZ in the pka1Δ or mal3Δ strains was suppressed by the overexpression of the Mal3 CH domain (1–143), Mal3 CH domain with the coiled-coil domain (1–197), or full-length Mal3. Overexpression of EB1 orthologs from Saccharomyces cerevisiae, Arabidopsis thaliana, Mus musculus, or Homo sapiens suppressed the TBZ-sensitive phenotype in the pka1Δ strains, indicating their conserved functions. These findings suggest that Pka1 and the microtubule binding of the Mal3 CH domain play a role in the maintenance of proper chromosome segregation.

Infigratinib Mediates Vascular Normalization, Impairs Metastasis, and Improves Chemotherapy in Hepatocellular Carcinoma.

The fibroblast growth factor (FGF) signaling cascade is a key signaling pathway in hepatocarcinogenesis. We report high FGF receptor (FGFR) expression in 17.7% (11 of 62) of hepatocellular carcinoma (HCC) models. Infigratinib, a pan‐FGFR inhibitor, potently suppresses the growth of high‐FGFR‐expressing and sorafenib‐resistant HCCs. Infigratinib inhibits FGFR signaling and its downstream targets, cell proliferation, the angiogenic rescue program, hypoxia, invasion, and metastasis. Infigratinib also induces apoptosis and vessel normalization and improves the overall survival of mice bearing FGFR‐driven HCCs. Infigratinib acts in synergy with the microtubule‐depolymerizing drug vinorelbine to promote apoptosis, suppress tumor growth, and improve the overall survival of mice. Increased expression levels of FGFR‐2 and FGFR‐3 through gene amplification correlate with treatment response and may serve as potential biomarkers for patient selection. Conclusion: Treatments with Infigratinib alone or in combination with vinorelbine may be effective in a subset of patients with HCC with FGFR‐driven tumors.

Primary Culture of Undifferentiated Pleomorphic Sarcoma: Molecular Characterization and Response to Anticancer Agents.

Undifferentiated pleomorphic sarcoma (UPS) is an aggressive mesenchymal neoplasm with no specific line of differentiation. Eribulin, a novel synthetic microtubule inhibitor, has shown anticancer activity in several tumors, including soft tissue sarcomas (STS). We investigated the molecular biology of UPS, and the mechanisms of action of this innovative microtubule-depolymerizing drug. A primary culture from a patient with UPS was established and characterized in terms of gene expression. The activity of eribulin was also compared with that of other drugs currently used for STS treatment, including trabectedin. Finally, Western blot analysis was performed to better elucidate the activity of eribulin. Our results showed an upregulation of epithelial mesenchymal transition-related genes, and a downregulation of epithelial markers. Furthermore, genes involved in chemoresistance were upregulated. Pharmacological analysis confirmed limited sensitivity to chemotherapy. Interestingly, eribulin exhibited a similar activity to that of standard treatments. Molecular analysis revealed the expression of cell cycle arrest-related and pro-apoptotic-related proteins. These findings are suggestive of aggressive behavior in UPS. Furthermore, the identification of chemoresistance-related genes could facilitate the development of innovative drugs to improve patient outcome. Overall, the results from the present study furnish a rationale for elucidating the role of eribulin for the treatment of UPS.

Division Plane Orientation Defects Revealed by a Synthetic Double Mutant Phenotype.

TANGLED1 (TAN1) and AUXIN-INDUCED-IN-ROOTS9 (AIR9) are microtubule-binding proteins that localize to the division site in plants. Their function in Arabidopsis (Arabidopsis thaliana) remained unclear because neither tan1 nor air9 single mutants have a strong phenotype. We show that tan1 air9 double mutants have a synthetic phenotype consisting of short, twisted roots with disordered cortical microtubule arrays that are hypersensitive to a microtubule-depolymerizing drug. The tan1 air9 double mutants have significant defects in division plane orientation due to failures in placing the new cell wall at the correct division site. Full-length TAN1 fused to yellow fluorescent protein, TAN1-YFP, and several deletion constructs were transformed into the double mutant to assess which regions of TAN1 are required for its function in root growth, root twisting, and division plane orientation. TAN1-YFP expressed in tan1 air9 significantly rescued the double mutant phenotype in all three respects. Interestingly, TAN1 missing the first 126 amino acids, TAN1-ΔI-YFP, failed to rescue the double mutant phenotype, while TAN1 missing a conserved middle region, TAN1-ΔII-YFP, significantly rescued the mutant phenotype in terms of root growth and division plane orientation but not root twisting. We use the tan1 air9 double mutant to discover new functions for TAN1 and AIR9 during phragmoplast guidance and root morphogenesis.

Abstract 3247: Discovery of a novel drug that affects centrosome clustering

Abstract Our goal is to identify molecular regulators of a mechanism that occurs uniquely in cancer cells, and to develop a selective anti-cancer drug. Most chemotherapies are non-selective, causing severe side-effects. In additions, cancers often develop resistance to some of the more commonly used chemotherapies. To expand the repertoire of available drugs, and to design drugs that are selective, we need to identify molecules that regulate the physiological changes that occur primarily in cancer cells. For example, cancer cells in many hard-to-treat cancers have aberrant centrosomes, which may be supernumerary or fragmented. During mitosis, these aberrant centrosomes must cluster to form bipolar spindles for successful division. Thus, targeting a process like centrosome clustering is ideal, since it is not necessary in healthy cells. We synthesized a small, stable scaffold with amenability to structure-activity-relationship studies, and found several analogues with IC50 values < 50 nM, depending on the cancer cell line. Preliminary tests showed that these compounds prevent tumors from forming and/or cause their regression in vitro. We performed cell biological studies to characterize their mechanism of action. In several different types of cancer cells, these compounds cause mitotic arrest and centrosome declustering at concentrations where they have little affect on non-cancerous cells. Live imaging revealed that within minutes of adding the compounds to HeLa cells expressing GFP-tagged tubulin, we observed rapid microtubule depolymerization and centrosome declustering. After washing out the compound, microtubule polymerization recovered, but the mitotic spindles were multipolar. Adding similar concentrations of Nocodazole, a microtubule-depolymerizing drug, also caused rapid microtubule depolymerization, but after washing out the drug, the spindles were bipolar. We are in the process of identifying the molecular target of these compounds to provide crucial insight to the mechanism governing centrosome clustering, and are continuing to perform SAR studies to obtain compounds with higher efficacy and selectivity. Citation Format: Dilan B. Jaunky, Kevin Larocque, Javier Porro Suardiaz, Dan Yang, Emma J. Furze, Pat Forgione, Alisa Piekny. Discovery of a novel drug that affects centrosome clustering [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3247. doi:10.1158/1538-7445.AM2017-3247

Uner Tan syndrome caused by a homozygous TUBB2B mutation affecting microtubule stability.

The integrity and dynamic properties of the microtubule cytoskeleton are indispensable for the development of the mammalian brain. Consequently, mutations in the genes that encode the structural component (the α/β-tubulin heterodimer) can give rise to severe, sporadic neurodevelopmental disorders. These are commonly referred to as the tubulinopathies. Here we report the addition of recessive quadrupedalism, also known as Uner Tan syndrome (UTS), to the growing list of diseases caused by tubulin variants. Analysis of a consanguineous UTS family identified a biallelic TUBB2B mutation, resulting in a p.R390Q amino acid substitution. In addition to the identifying quadrupedal locomotion, all three patients showed severe cerebellar hypoplasia. None, however, displayed the basal ganglia malformations typically associated with TUBB2B mutations. Functional analysis of the R390Q substitution revealed that it did not affect the ability of β-tubulin to fold or become assembled into the α/β-heterodimer, nor did it influence the incorporation of mutant-containing heterodimers into microtubule polymers. The 390Q mutation in S. cerevisiae TUB2 did not affect growth under basal conditions, but did result in increased sensitivity to microtubule-depolymerizing drugs, indicative of a mild impact of this mutation on microtubule function. The TUBB2B mutation described here represents an unusual recessive mode of inheritance for missense-mediated tubulinopathies and reinforces the sensitivity of the developing cerebellum to microtubule defects.

Linking Nucleoporins, Mitosis, and Colon Cancer

Suppression of a nuclear pore protein Nup358/RanBP2 is linked to mitotic cell death, but the clinical relevance of this link is unknown. In a recent issue of Cell, Vecchione etal. (2016) show that in approximately 10% of BRAF-like colorectal cancer (CC) patients, Nup358/RanBP2 is critical for survival. Treatment with vinorelbine, a microtubule-depolymerizing drug that inhibits mitosis, might be a potential treatment for theseCCs.

Eribulin mesylate: mechanism of action of a unique microtubule-targeting agent.

Eribulin mesylate (eribulin), an analogue of the marine natural product halichondrin B, is a microtubule-depolymerizing drug that has utility in the treatment of patients with breast cancer. Clinical trial results have demonstrated that eribulin treatment provides a survival advantage to patients with metastatic or locally advanced breast cancer previously treated with an anthracycline and a taxane. Furthermore, a pooled analysis of two pivotal phase III trials has demonstrated that eribulin also improves overall survival in several patient subgroups, including in women with HER2-negative disease and triple-negative breast cancer. This review covers the preclinical research that led to the clinical testing and approval of eribulin, as well as subsequent research that was prompted by distinct and unexpected effects of eribulin in the clinic. Initial studies with halichondrin B demonstrated unique effects on tubulin binding that resulted in distinct microtubule-dependent events and antitumor actions. Consistent with the actions of the natural product, eribulin has potent microtubule-depolymerizing activities and properties that distinguish it from other microtubule-targeting agents. Here, we review new results that further differentiate the effects of eribulin from other agents on peripheral nerves, angiogenesis, vascular remodeling, and epithelial-to-mesenchymal transition. Together, these data highlight the distinct properties of eribulin and begin to delineate the mechanisms behind the increased survival benefit provided by eribulin for patients.

Tethering sister centromeres to each other suggests the spindle checkpoint detects stretch within the kinetochore.

The spindle checkpoint ensures that newly born cells receive one copy of each chromosome by preventing chromosomes from segregating until they are all correctly attached to the spindle. The checkpoint monitors tension to distinguish between correctly aligned chromosomes and those with both sisters attached to the same spindle pole. Tension arises when sister kinetochores attach to and are pulled toward opposite poles, stretching the chromatin around centromeres and elongating kinetochores. We distinguished between two hypotheses for where the checkpoint monitors tension: between the kinetochores, by detecting alterations in the distance between them, or by responding to changes in the structure of the kinetochore itself. To distinguish these models, we inhibited chromatin stretch by tethering sister chromatids together by binding a tetrameric form of the Lac repressor to arrays of the Lac operator located on either side of a centromere. Inhibiting chromatin stretch did not activate the spindle checkpoint; these cells entered anaphase at the same time as control cells that express a dimeric version of the Lac repressor, which cannot cross link chromatids, and cells whose checkpoint has been inactivated. There is no dominant checkpoint inhibition when sister kinetochores are held together: cells expressing the tetrameric Lac repressor still arrest in response to microtubule-depolymerizing drugs. Tethering chromatids together does not disrupt kinetochore function; chromosomes are successfully segregated to opposite poles of the spindle. Our results indicate that the spindle checkpoint does not monitor inter-kinetochore separation, thus supporting the hypothesis that tension is measured within the kinetochore.

31 PROTOCOL OPTIMIZATION AND EVALUATION OF MATURATION PROMOTING FACTOR AND MITOGEN-ACTIVATED PROTEIN KINASE ACTIVITIES IN BOVINE CYTOPLASTS OBTAINED BY CHEMICAL ENUCLEATION TECHNIQUES

Chemical enucleation using microtubule-depolymerizing drugs is an attractive procedure to simplify the enucleation process in nuclear transfer. The aim of this study was to optimize chemically assisted (CA) and chemically induced (CI) enucleation protocols using metaphase II (MII) and pre-activated bovine oocytes, respectively, and to evaluate the activity of maturation promoting factor (MPF) and mitogen-activated protein kinase (MAPK) in cytoplasts generated by these techniques. Initially, we determined the shortest effective treatment of MII and activated oocytes with 0.05 μg mL–1 demecolcine. Bovine oocytes in vitro matured (IVM) for 19 h (MII) or activated artificially with 5 μM ionomycin (5 min) and 10 μg mL–1 cycloheximide (5 h) after 26 h IVM were treated with demecolcine and samples were collected at 0, 0.25, 0.5, 1.0, 1.5, and 2.0 h of treatment. Oocytes were then stained with 10 μg mL–1 Hoechst 33342 and the protrusion or enucleation rates were determined. Next, we evaluated histone H1 and myelin basic protein (MBP) kinases, reflecting MPF and MAPK activities, respectively, in oocytes obtained from these treatments, and for that we used the method described by Kubelka et al. (2000 Biol. Reprod. 62, 292–302). Protrusion and enucleation rates were evaluated by the chi-squared (χ2) test, and MPF and MAPK activities were submitted to ANOVA and Tukey's test at 5% significance. For MII oocytes, effects of demecolcine were observed as early as 15 min, with a significant difference (P < 0.05) between control (12/112, 10.7%) and treated (33/114, 28.9%) groups in relation to protrusion rates. The largest number of protrusions was observed after 1.0 h of treatment (control: 15/113, 13.3%a; treated: 45/111, 40.5%b). In pre-activated oocytes, effects of demecolcine were also observed after 15 min, and in both techniques there were no significant differences between groups treated with demecolcine for 1.0, 1.5, or 2.0 h (CA: 40.5 to 52.5% of protrusion; CI: 35.2 to 46.7% of enucleation). In contrast to previous reports in which high concentrations of demecolcine for CA enucleation increased MPF activity, we observed no alterations in the activity of this factor at a demecolcine concentration of 0.05 μg mL–1. Activity of MAPK also did not differ significantly between the control and treated groups throughout evaluation. In the CI technique, a significant difference in MPF activity was observed after 0.5 h (70.3%) and 2.0 h of activation (39.1%), considering that the activity was 100% at the beginning of the evaluation. However, we observed no significant difference between the control and treated groups at any of the time points studied, as verified for MAPK activity. The exact effect of MPF on the nucleus in mammals is not well established. We believe that the use of low concentrations of demecolcine for short periods is less damaging to embryonic development and, until we have a better understanding of the effect of these kinases on the transferred nucleus, we recommend its use for chemical enucleation protocols in bovine. Financial support: FAPESP 2010/20744-6 and 2011/12983-3.

Colchicine-induced rhabdomyolysis following a concomitant use of clarithromycin in a haemodialysis patient with familial Mediterranean fever.

Sir, Renal amyloidosis secondary to familial Mediterranean fever (FMF) is one of the common causes of nephrotic syndrome and chronic kidney disease in Turkey [1]. Colchicine is a microtubule-depolymerizing drug widely used for the treatment of gout which may prevent the development of secondary amyloidosis. In end-stage renal disease (ESRD), although the dose of colchicine is suggested to be reduced, there is limited information on efficacy and safety [2]. Inappropriately high doses or co-prescription of CYP3A4 or P-glycoprotein inhibitors have been reported to cause serious adverse effects, including death [3–7]. Herein, we present a case of colchicine-induced rhabdomyolysis after using clarithromycin, a macrolide antibiotic and a potent inhibitor of CYP3A4 and P-glycoprotein ABCB1. A 40-year old male patient on maintenance haemodialysis was admitted with recently developed fatigue, anorexia, headache, dizziness, diffuse myalgia and gastrointestinal symptoms including epigastric pain, diarrhoea and bloating. He presented with FMF in childhood (with frequent attacks of fever and abdominal pain with arthritis) and had developed secondary amyloidosis 10 years previously and ESRD 6 months previously. His long-term medication was colchicine 0.5 mg twice daily, doxazosin 4 mg once daily and 40 mg furosemide on non-dialysis days and sevelamer 1600 mg three times a day. A further history revealed that the patient had recently been diagnosed with acute sinusitis and was prescribed clarithromycin 500 mg twice daily in another hospital a week beofore. On his initial examination the patient's blood pressure was 100/60 mmHg, he was in a euvolaemic state and had no significant finding except diffuse tenderness, but there was no rebound on abdominal palpation. In his biochemistry, his creatine kinase (CK)(5732 U/L- 9035 U/L) and transaminase levels (ALT:110 U/L, AST:309 U/L) were elevated and raised upon follow-up. Due to high CK and transaminases, colchicine-induced rhabdomyolysis was diagnosed and colchicine discontinued. The patient's pain subsided 3 days after discontinuation of colchicine. An EMG was performed because of high levels of CK to exclude the diseases causing myopathie. During outpatient follow-up, the patient's transaminases and CK returned to normal levels and his EMG was normal. After complete recovery, the patient started colchicine 0.5 mg/day again without recurrence of symptoms.

A checkpoint escape

In most dividing cells, the spindle assembly checkpoint (SAC), which monitors kinetochore–microtubule interactions, signals to prevent anaphase onset until all chromosomes are correctly attached to the metaphase spindle. Here, Shao et al. reveal that Xenopus laevis oocytes do not respond to a SAC. The authors first developed a karyotyping technique that allows the analysis of meiotic chromosomes after nuclear envelop breakdown. When treating oocytes during meiosis I with microtubule-depolymerizing drugs that destroyed the spindle, they observed that homologous chromosomes could still divide into single pairs of chromatids, and on time. This suggested that oocytes could progress to meiosis II and arrest at metaphase II, as normal, without a spindle. Similarly, defective oocytes exhibiting monopolar spindles proceeded through monopolar anaphase without delay, indicating that, in X. laevis, the metaphase-to-anaphase transition is not under SAC regulation.

CLASP Interacts with Sorting Nexin 1 to Link Microtubules and Auxin Transport via PIN2 Recycling in Arabidopsis thaliana

Polarized movement of auxin generates concentration gradients within plant tissues to control cell division patterns and growth direction by modulating microtubule organization. In this study, we identify a reverse mechanism, wherein microtubules influence polar auxin transport. We show that the microtubule-associated protein CLASP interacts with the retromer component sorting nexin 1 (SNX1) to mediate an association between endosomes and microtubules. clasp-1 null mutants display aberrant SNX1 endosomes, as do wild-type plants treated with microtubule-depolymerizing drugs. Consistent with SNX1's role in trafficking of the auxin efflux carrier PIN-FORMED2 (PIN2), clasp-1 mutant plants have enhanced PIN2 degradation, and PIN2 movement to lytic vacuoles is rapidly induced by depolymerization of microtubules. clasp-1 mutants display aberrant auxin distribution and exhibit numerous auxin-related phenotypes. In addition to mechanistically linking auxin transport and microtubules, our data identify a ubiquitous endosome-microtubule association in plants.