Inhibition Of Microtubule Formation Research Articles

Mechanisms of mitotic inhibition in human aorta endothelial cells: Molecular and morphological in vitro spectroscopic studies

Mitotic inhibitors are drugs commonly used in chemotherapy, but their nonspecific and indiscriminate distribution throughout the body after intravenous administration can lead to serious side effects, particularly on the cardiovascular system. In this context, our investigation into the mechanism of the cytotoxic effects on endothelial cells of mitotic inhibitors widely used in cancer treatment, such as paclitaxel (also known as Taxol) and Vinca alkaloids, holds significant practical implications. Understanding these mechanisms can lead to more targeted and less harmful cancer treatments.Human aorta endothelial cells (HAECs) were incubated with selected mitotic inhibitors in a wide range of concentrations close to those in human plasma during anticancer therapy. The analysis of single cells imaged by Raman spectroscopy allowed for visualization of the nuclear, cytoplasmic, and perinuclear areas to assess biochemical changes induced by the drug’s action. The results showed significant changes in the morphology and molecular composition of the nucleus. Moreover, an effect of a given drug on the cytoplasm was observed, which can be related to its mechanism of action (MoA). Raman data supported by fluorescence microscopy measurements identified unique changes in DNA form and proteins and revealed drug-induced inflammation of endothelial cells.The primary goal of mitotic inhibitors is based on the impairment of tubulin formation and the inhibition of the mitosis process. While all three drugs affect microtubules and disrupt cell division, they do so through different MoA, i.e., Vinca alkaloids inhibit microtubule formation, whereas paclitaxel stabilizes microtubules. To sum up, the work shows how a specific drug can interact with endothelial cells.

Frentizole, a Nontoxic Immunosuppressive Drug, and Its Analogs Display Antitumor Activity via Tubulin Inhibition.

Antimitotic agents are one of the more successful types of anticancer drugs, but they suffer from toxicity and resistance. The application of approved drugs to new indications (i.e., drug repurposing) is a promising strategy for the development of new drugs. It relies on finding pattern similarities: drug effects to other drugs or conditions, similar toxicities, or structural similarity. Here, we recursively searched a database of approved drugs for structural similarity to several antimitotic agents binding to a specific site of tubulin, with the expectation of finding structures that could fit in it. These searches repeatedly retrieved frentizole, an approved nontoxic anti-inflammatory drug, thus indicating that it might behave as an antimitotic drug devoid of the undesired toxic effects. We also show that the usual repurposing approach to searching for targets of frentizole failed in most cases to find such a relationship. We synthesized frentizole and a series of analogs to assay them as antimitotic agents and found antiproliferative activity against HeLa tumor cells, inhibition of microtubule formation within cells, and arrest at the G2/M phases of the cell cycle, phenotypes that agree with binding to tubulin as the mechanism of action. The docking studies suggest binding at the colchicine site in different modes. These results support the repurposing of frentizole for cancer treatment, especially for glioblastoma.

Fluchloralin induces developmental toxicity in heart, liver, and nervous system during early zebrafish embryogenesis

The zebrafish is a prominent vertebrate model popularly used for toxicity testing because of its rapid development and transparent embryos. Fluchloralin, a dinitroaniline herbicide used to control weeds, inhibits microtubule formation and cell division. The structurally homologous substances ethalfluralin and pendimethalin, which belong to the dinitroaniline family, were found to be genotoxic and to exert developmental toxicity via mitochondrial dysfunction in a zebrafish model. To date, developmental toxicity of fluchloralin in zebrafish has not been reported. In the present study, we identified morphological changes in developing zebrafish, including decreased survival rate and body length, and increased yolk sac edema. In dose-dependent response to fluchloralin exposure, inhibition of neurogenesis in the spinal cord and motor neuron defects were observed in transgenic zebrafish models (olig2:dsRed). Zebrafish exposed to fluchloralin also displayed organ dysfunction in the heart, liver, and pancreas in cmlc2:dsRed and lfabp:dsRed;elastase:GFP transgenic models. Fluchloralin increased cell death in the brain by promoting apoptosis, visualized via acridine orange staining, and by activating apoptosis signaling proteins, including cytochrome c1, zBax, and Bcl-XL. This study provides novel evidence supporting the necessity of controlling pollutants in aquatic environments.

Abstract 1990: Targeting cancer stem cells is an answer to the curing lung cancer

Abstract Lung cancer is the leading cause of cancer-related mortality worldwide. Mainly, non-small cell lung cancer (NSCLC, about 80% of cases) and small cell lung cancer (SCLC, about 20% of cases) are the major types of lung cancer. Adenocarcinoma (30-50%) and squamous cell carcinoma (30%) are the major subtypes of NSCLC. Even though there are considerable advancements in the drugs against lung cancer, the 5-year survival rate is meager. Unfortunately, the drug resistance for currently available anticancer drugs results in a 5-year survival of only 5%. The remarkable lung cancer recurrence despite definitive local and/or systemic therapy indicate that residual cancer stem cells (CSCs) possess tumorigenic potential. Therefore, drugs targeting the CSCs and killing them quickly can cure lung cancer without any relapse and improve the 5-year survival rate. Herein, we present the application of the recently reported ID-Checker technology (Journal of Medicinal Chemistry 2022 65 (19), 12883-12894) for killing lung cancer stem cells (LCSC). The ID-Checker that is decorated with the glucose tag allows highly specific absorption and macroscale delivery of anticancer drugs to the LCSC through the GLUT channels. ID-Checker inhibits the microtubule formation resulting in the cell cycle arrest in the G2/M phase followed by apoptosis. The inhibition of microtubule formation by ID-Checker also leads to the inhibition of GLUT channel deployment in the LCSC. Thus, the glucose-deprived LCSC undergoes apoptosis within 72 hours of ID-Checker treatment. Hence, we believe the ID-Check technology presented here can kill the LCSC and completely cure lung cancer. Citation Format: Keum-Soo Song, Satish Balasaheb Nimse, Taisun Kim. Targeting cancer stem cells is an answer to the curing lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1990.

BI-D1870 Induces Mitotic Dysfunction and Apoptosis in Neuroblastoma by Regulating the PI3K-Akt-mTORC1 Signal Axis

Introduction: Neuroblastoma (NB) is one of the most common extracranial solid malignant tumors in children. The 5-year survival rate of high-risk or refractory NB is less than 50%. Therefore, developing new effective therapeutics for NB remains an urgent challenge. Materials and Methods: Based on the NB dataset TARGET-NBL in the TCGA database, the prognosis-related genes were analyzed using univariate cox regression (p < 0.01). The protein network interaction of prognostic genes was analyzed using STRING to obtain 150 hub genes with HR > 1 and 150 hub genes with HR < 1. The Connectivity Map database was used to predict a therapeutic drug: BI-D1870, a ribosomal S6 kinase inhibitor. The inhibitory effect of BI-D1870 on NB was investigated through in vivo and in vitro experiments, and its inhibitory mechanism was explored. Results: Both the in vivo and in vitro experiments showed that BI-D1870 could inhibit tumor proliferation and induce tumor apoptosis. Furthermore, we proved that BI-D1870 caused G2/M phase arrest and mitosis damage in cells. RNA-seq of cells showed that BI-D1870 may inhibit the growth of NB by inhibiting the PI3K-Akt-mTOR axis. Western blot and immunofluorescence testing showed that BI-D1870 inhibited the PI3K-Akt-mTORC1 signal pathway to regulate the phosphorylation of RPS6 and 4E BP1 proteins, inhibit protein translation, and inhibit microtubule formation, thus preventing mitotic proliferation and inducing apoptosis. Conclusions: This study provides strong support that BI-D1870 may be a potential adjuvant therapy for NB.

Recent Advances of Tubulin Inhibitors Targeting the Colchicine Binding Site for Cancer Therapy.

Cancer accounts for numerous deaths each year, and it is one of the most common causes of death worldwide, despite many breakthroughs in the discovery of novel anticancer candidates. Each new year the FDA approves the use of new drugs for cancer treatments. In the last years, the biological targets of anticancer agents have started to be clearer and one of these main targets is tubulin protein; this protein plays an essential role in cell division, as well as in intracellular transportation. The inhibition of microtubule formation by targeting tubulin protein induces cell death by apoptosis. In the last years, numerous novel structures were designed and synthesized to target tubulin, and this can be achieved by inhibiting the polymerization or depolymerization of the microtubules. In this review article, recent novel compounds that have antiproliferation activities against a panel of cancer cell lines that target tubulin are explored in detail. This review article emphasizes the recent developments of tubulin inhibitors, with insights into their antiproliferative and anti-tubulin activities. A full literature review shows that tubulin inhibitors are associated with properties in the inhibition of cancer cell line viability, inducing apoptosis, and good binding interaction with the colchicine binding site of tubulin. Furthermore, some drugs, such as cabazitaxel and fosbretabulin, have been approved by FDA in the last three years as tubulin inhibitors. The design and development of efficient tubulin inhibitors is progressively becoming a credible solution in treating many species of cancers.

Evaluation of the mechanism of action of albendazole on adult rat lungworm (Angiostrongyluscantonensis)

Albendazole is considered the anthelmintic of choice for the management of rat lungworm disease (neuroangiostrongyliasis), due to its broad spectrum of nematocidal activity and its ability to cross the blood-brain barrier. Albendazole binds to β-tubulins, preventing their polymerization into microtubules, thereby corrupting the cascade of cell division at metaphase, which ultimately leads to the death of individual cells and eventually the death of the parasite. Inhibition of microtubule formation will also hinder the axoplasmic transport system, affecting the neuronal activities of the parasite. While this mechanism has been explicated in other parasitic and non-parasitic nematodes, it has never been evaluated in Angiostrongylus cantonensis. This study evaluates the antimitotic effects of albendazole sulphoxide (active metabolite) on the microtubules of adult A. cantonensis using the tubulin polymerization assay and measures its effects on worm viability using the colorimetric MTT assay. Three different concentrations of albendazole (62.5 μM, 250 μΜ, and 1 mM) were evaluated. We saw a statistically significant dose-dependent reduction in the band intensity of polymerized tubulins (or microtubules) (P = 0.019), suggesting that albendazole imparts its antimitotic effect in a dose-dependent manner. Similarly, our MTT assay showed a dose-dependent decrease in formazan intensity (proportional to cell viability), suggesting that the rate of nematocidal activity of albendazole is also proportional to its concentration. In compiling the results from both these experiments, a correlation between the microtubule assembly and worm viability is evident.

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Abstract 6206: Discerning vasculature destruction caused by novel vascular disrupting agents examined by optical and multispectral optoacoustic imaging

Abstract Tumor growth requires a functioning vascular network and offers an attractive therapeutic target. The invasive proliferating neovasculature of tumors lacks pericyte support and exhibits increased permeability thereby offering a unique, potentially selective target for anticancer therapy. Most vascular disrupting agents (VDAs) inhibit microtubule formation, causing rapid morphological changes in endothelia cells resulting in dramatically increased vessel permeability, cellular detachment, vessel occlusion, and vessel wall damage [doi: 10.3390/molecules26092551]. Highly vascular kidney tumors are expected to be particularly sensitive to VDAs. We are evaluating OXi8007, a water soluble indole phosphate pro-drug derivative of the first generation VDA combretastatin. The prodrug OXi8007 is stable in saline but releases active OXi8006 is the presence of alkaline phosphate. Preliminary pharmacokinetic data benchmarked using 13C-labeled OXi8006 and OXi8007 show rapid vascular clearance and conversion to the glucuronide conjugate in vivo. Doses up to 700 mg/kg were tolerated by mice. Luciferase-expressing RENCA tumor cells were implanted orthotopically in the right kidney capsule of syngeneic BALB/c mice. Bioluminescence Imaging (BLI) was applied weekly to assess tumor growth and acute response to OXi8007. Following addition of luciferin substrate subcutaneously BLI signal from the tumor reached a maximum within 10 minutes. When BLI was repeated 4 hrs after 250 or 350 mg/kg OXi8007 IP, the generated signal was &amp;lt;1% of baseline signal indicating acute vascular disruption. Multispectral Optoacoustic Tomography (MSOT) revealed matching acute changes in tumor vascular oxygenation and additionally showed distinct heterogeneity. An oxygen gas breathing challenge indicated lower oxygen saturation in the tumor following treatment. Histology showed massive hemorrhage following both doses. BLI signal recovered after 1 to 2 days and repeated administration of OXi8007 again caused acute vascular shutdown. These results match previous observations in breast tumor models [doi: 10.1016/j.canlet.2015.08.021] indicating opportunities for therapeutic application. Citation Format: Hashini I. Wanniarachchi, Regan Schuetze, Lorena Arango, Khagendra Hamal, Graham J. Carlson, Cyprian Pavlich, Yuling Deng, Mary L. Trawick, Kevin G. Pinney, Li Liu, Ralph P. Mason. Discerning vasculature destruction caused by novel vascular disrupting agents examined by optical and multispectral optoacoustic imaging [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6206.

Inhibition of microtubule assembly and cytotoxic effect of graphene oxide on human colorectal carcinoma cell HCT116

Nanomaterials, such as graphene oxide (GO), are increasingly being investigated for their suitability in biomedical applications. Tubulin is the key molecule for the formation of microtubules crucial for cellular function and proliferation, and as such an appealing target for developing anticancer drug. Here we employ biophysical techniques to study the effect of GO on tubulin structure and how the changes affect the tubulin/microtubule assembly. GO disrupts the structural integrity of the protein, with consequent retardation of tubulin polymerization. Investigating the anticancer potential of GO, we found that it is more toxic to human colon cancer cells (HCT116), as compared to human embryonic kidney epithelial cells (HEK293). Immunocytochemistry indicated the disruption of microtubule assembly in HCT116 cells. GO arrested cells in the S phase with increased accumulation in Sub-G1 population of cell cycle, inducing apoptosis by generating reactive oxygen species (ROS) in a dose- and time-dependent manner. GO inhibited microtubule formation by intervening into the polymerization of tubulin heterodimers both in vitro and ex vivo, resulting in growth arrest at the S phase and ROS induced apoptosis of HCT116 colorectal carcinoma cells. There was no significant harm to the HEK293 kidney epithelial cells used as control. Our report of pristine GO causing ROS-induced apoptosis of cancer cells and inhibition of tubulin-microtubule assembly can be of interest in cancer therapeutics and nanomedicine.

Machine learning tools can now help with improving Taxol production in plant cell cultures

Chemotherapeutic intervention for cancer care is an important step. One of the most effective chemotherapy agents in use today is Paclitaxel (PTX), sold under the common name Taxol and Oxanol. Due to its ability to inhibit microtubule formation in cells, PTX is effective at all stages of the cancer and is FDA approved for treatment of many types of cancer (ovarian cancer, esophageal cancer, breast cancer, lung cancer, Kaposi sarcoma, cervical cancer, and pancreatic cancer). PTX is a plant alkaloid in the taxane family of compounds obtained from bark of the Pacific Yew tree (Taxus brevifolia) [1]. Adequate market supply of PTX has remained a challenge, as paclitaxel represents only a minor proportion of the total taxoid content of the Taxus species. Over the years, research into finding an alternate to cutting down Yew trees for PTX harvesting has been on the forefront. It is estimated that up to 60 trees may need to be harvested for the treatment of one patient.

Abstract 3048: Murine-derived colorectal cancer organoid culture high-throughput drug screening to identify novel combinations targeting PIK3CA mutant cancers

Abstract Introduction: Colorectal cancer (CRC) treatment is becoming increasingly individualized as clinical mutation profiling is a standard of care. Oncogenic mutations of the PIK3CA gene induce a constitutively active phosphatidylinositol 3-kinase with downstream effects that promote tumor initiation and progression. Effective clinical therapeutic strategies targeting this subtype of colorectal cancer have remained elusive. Here we develop a high-throughput organoid drug screening platform to examine combination strategies targeting PIK3CA mutant CRC. Methods: Murine-derived CRC organoids (MDCOs) were derived from a transgenic mouse model of PIK3CA and APC mutated colorectal cancer (F1 (FVBxB6) Apcfl/+ Pik3caH1047R), matured for 48 hours and plated in 96 well plates. A total of 96 compounds chosen from clinically available anticancer drugs and select drugs in clinical development were analyzed for their efficacy in reducing organoid growth on their own, as well as in combination with copanlisib (PI3K/MTOR inhibitor) over 48 hours. Select treatments were confirmed using our standard 24 well plate assay. Additionally, irinotecan (SN38; topoisomerase-I inhibitor), panitumumab (EGFR inhibitor), and olaparib (PARP inhibitor) were investigated alone and in combination with copanlisib based on clinical use for this population or recent promising clinical trial data. Results: SN38, olaparib, and panitumumab yielded insignificant changes in spheroid size in the combination regimen compared to copanlisib alone (median relative change in organoid diameter - copanlisib -8%, p&amp;lt;0.001; SN38 combo -8%, p=0.5; olaparib combo -13%, p=0.6; and panitumumab combo 1%, p=0.9). The MDCOs were successfully utilized for the high-throughput drug screen. Differential sensitivity was identified using change in organoid diameter without the need for reagents or dyes which could complicate the screening method. Of the 96 compounds investigated in the high-throughput screen, 12 resulted in enhanced treatment effect when combined with copanlisib. Three of these 12 compounds inhibit microtubule formation, and others inhibit components of the RAS pathway. Two of these drug combinations were examined further and significant treatment responses were confirmed. Conclusion: Advances in the understanding of the molecular basis of cancer allow for a more precise approach to cancer treatment. High-throughput techniques are needed to better identify novel treatment strategies for molecular subtypes. Here we describe a label free murine-derived cancer organoid method to screen drugs for combination treatment strategies. Citation Format: Alyssa K. DeZeeuw, Rebecca A. DeStefanis, Gioia Sha, Cheri A. Pasch, Linda Clipson, Dustin A. Deming. Murine-derived colorectal cancer organoid culture high-throughput drug screening to identify novel combinations targeting PIK3CA mutant cancers [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3048.

Discovery of dihydrofuranoallocolchicinoids - Highly potent antimitotic agents with low acute toxicity.

Two series of heterocyclic colchicinoids bearing β-methylenedihydrofuran or 2H-pyran-2-one fragments were synthesized by the intramolecular Heck reaction. Methylenedihydrofuran compounds 9a and 9h were found to be the most cytotoxic among currently known colchicinoids, exhibiting outstanding antiproliferative activity on tumor cell lines in picomolar (0.01-2.1nM) range of concentrations. Compound 9a potently and substoichiometrically inhibits microtubule formation invitro, being an order of magnitude more active in this assay than colchicine. Derivatives 9a and 9h revealed relatively low acute toxicity in mice (LD50≥10mg/kg i.v.). The X-Ray structure of colchicinoid 9a bound to tubulin confirmed interaction of this compound with the colchicine binding site of tubulin.

Fluorinated derivatives of 2-phenyl-3-hydroxy-4(1H)-quinolinone as tubulin polymerization inhibitors

We have synthesized a series of 2-phenyl-3-hydroxy-4(1H)-quinolinone derivatives substituted with one or more fluorine atoms on the quinolone backbone as well as on phenyl ring. The derivatives bearing more fluorine atoms were subjected to modification by nucleophilic substitutions by thiophenol, morpholine, and piperazine derivative. We have tested the prepared compounds in cytotoxic activity assay against cancer cell lines. Four derivatives exhibited micromolar values of IC50 against some of the cancer cell lines, and we have subjected them to cell cycle analysis on CCRF-CEM. Moreover, most active 7-fluoro-3-hydroxy-2-phenyl-6-(phenylthio)quinolin-4(1H)-one inhibits mitosis progression. Cell cycle analysis, in vitro tubulin polymerization assay, and tubulin imaging in cells indicated that the anticancer activity of thiophenol derivative is associated with its ability to inhibit microtubule formation.

Benomyl, a benzimidazole fungicide, induces oxidative stress and apoptosis in neural cells

Fungicides are used in the agricultural sector against the harmful action of fungi, however they are potential toxic agents for the environment and the living organisms. Benomyl is a widely encountered benzimidazole fungicide that exerts its toxicity via inhibiting microtubule formation in the nervous system and the male reproductive and endocrine systems, whilst it is a known teratogen. Since toxic effects of benomyl and its molecular mechanisms are not fully understood, we aimed to detect its neurotoxic potential via evaluating cytotoxicity, oxidative stress and apoptosis in SH-SY5Y cell line. The cells were incubated with benomyl in a concentration range between 1 and 6 μM for 24 h. Our results indicated a concentration-dependent enhancement of reactive oxygen species measured through flow cytometry and DNA damage evaluated via the comet assay. Additionally, it induced apoptosis in all tested concentrations. According to the findings of the present study, benomyl is a xenobiotic, which it appears to exert its toxic action via a redox-related mechanism that, finally, induces cell apoptosis and death. We believe that this study will offer further insight in the toxicity mechanism of benomyl, although further studies are recommended in order to elucidate these mechanisms in the molecular level.

In-Vitro Investigation of the Cytotoxic and Genotoxic Effects of Benzimidazole Group Pesticides Benomyl and Carbendazim

In-Vitro Investigation of the Cytotoxic and Genotoxic Effects of Benzimidazole Group Pesticides Benomyl and Carbendazim. Fungicides are the most effective method to control fungal microorganisms which cause plant diseases. The benzimidazole group of fungicides acts by inhibiting microtubule formation.

Tau regulates the microtubule-dependent migration of glioblastoma cells via the Rho-ROCK signaling pathway.

The pathological significance of Tau (encoded by MAPT) in mechanisms driving cell migration in glioblastoma is unclear. By using an shRNA approach to deplete microtubule-stabilizing Tau in U87 cells, we determined its impact on cytoskeletal coordination during migration. We demonstrated here that the motility of these Tau-knockdown cells (shTau cells) was significantly (36%) lower than that of control cells. The shTau cells displayed a slightly changed motility in the presence of nocodazole, which inhibits microtubule formation. Such reduced motility of shTau cells was characterized by a 28% lower number of microtubule bundles at the non-adhesive edges of the tails. In accordance with Tau-stabilized microtubules being required for cell movement, measurements of the front, body and rear section displacements of cells showed inefficient tail retraction in shTau cells. The tail retraction was restored by treatment with Y27632, an inhibitor of Rho-ROCK signaling. Moreover, we clearly identified that shTau cells displayed relocation of the active phosphorylated form of p190-RhoGAP (also known as ARHGAP35), which inhibits Rho-ROCK signaling, and focal adhesion kinase (FAK, also known as PTK2) in cell bodies. In conclusion, our findings indicate that Tau governs the remodeling of microtubule and actin networks for the retraction of the tail of cells, which is necessary for effective migration.

Repositioning of Anti-parasitic Drugs in Cyclodextrin Inclusion Complexes for Treatment of Triple-Negative Breast Cancer.

Drug repositioning refers to the identification of new therapeutic indications for drugs already approved. Albendazole and ricobendazole have been used as anti-parasitic drugs for many years; their therapeutic action is based on the inhibition of microtubule formation. Therefore, the study of their properties as antitumor compounds and the design of an appropriate formulation for cancer therapy is an interesting issue to investigate. The selected compounds are poorly soluble in water, and consequently, they have low and erratic bioavailability. In order to improve their biopharmaceutics properties, several formulations employing cyclodextrin inclusion complexes were developed. To carefully evaluate the in vitro and in vivo antitumor activity of these drugs and their complexes, several studies were performed on a breast cancer cell line (4T1) and BALB/c mice. In vitro studies showed that albendazole presented improved antitumor activity compared with ricobendazole. Furthermore, albendazole:citrate-β-cyclodextrin complex decreased significantly 4T1 cell growth both in in vitro and in vivo experiments. Thus, new formulations for anti-parasitic drugs could help to reposition them for new therapeutic indications, offering safer and more effective treatments by using a well-known drug.

A novel gene, cilia flagella associated protein 44, encoding an enzyme cleaving FtsZ and tubulin contributes to the regulation of secretory pathway

We identified a novel gene, encoding the central region of the cilia and flagella associated protein 44 (Cfap44), that regulates trafficking of cellular components and morphology via the cleavage of cellular proteins (particularly β-tubulin). Although Cfap44 is registered in GenBank, the functions of both the central part and full-length protein are unknown except for a polymorphism associated with proprotein convertase 9 activity, the third gene of familiar hyper-cholesterolemia. In mice and humans, both unspliced and spliced RNAs were transcribed, and the spliced form was predominantly transcribed in the brain and embryonic tissues. In transfectants carrying this gene, various cellular processes such as cell division, transport of cellular components, and proteolytic processing of several proteins were found to be affected. The cleavage of β-tubulin was observed. A bacterial tubulin homolog, cell division protein FtsZ, was also cleaved in vivo and in vitro by the spliced form of Cfap44 product. Furthermore, the unspliced form showed proteolytic activity with low substrate specificity. Various biological activities of Cfap44 may be due to a direct effect of cleavage of β-tubulin inhibiting microtubule formation, or an indirect effect with the cross-recognition of the cleavage site between β-tubulin and other molecules.

The DNA replication protein Cdc6 inhibits the microtubule-organizing activity of the centrosome

The centrosome serves as a major microtubule-organizing center (MTOC). The Cdc6 protein is a component of the pre-replicative complex and a licensing factor for the initiation of chromosome replication and localizes to centrosomes during the S and G2 phases of the cfell cycle of human cells. This cell cycle-dependent localization of Cdc6 to the centrosome motivated us to investigate whether Cdc6 negatively regulates MTOC activity and to determine the integral proteins that comprise the pericentriolar material (PCM). Time-lapse live-cell imaging of microtubule regrowth revealed that Cdc6 depletion increased microtubule nucleation at the centrosomes and that expression of Cdc6 in Cdc6-depleted cells reversed this effect. This increase and decrease in microtubule nucleation correlated with the centrosomal intensities of PCM proteins such as γ-tubulin, pericentrin, CDK5 regulatory subunit-associated protein 2 (CDK5RAP2), and centrosomal protein 192 (Cep192). The regulation of microtubule nucleation and the recruitment of PCM proteins to the centrosome required Cdc6 ATPase activity, as well as a centrosomal localization of Cdc6. These results suggest a novel function for Cdc6 in coordinating centrosome assembly and function.