The differential distribution of acetylated and detyrosinated alpha-tubulin in the microtubular cytoskeleton and primary cilia of hyaline cartilage chondrocytes

Histone deacetylase 6 (HDAC6) is an emerging therapeutic target that is overexpressed in glioblastoma when compared to other HDACs. HDAC6 catalyzes the deacetylation of alpha-tubulin and mediates the disassembly of primary cilia, a process required for cell cycle progression. HDAC6 inhibition disrupts glioma proliferation, but whether this effect is dependent on tumor cell primary cilia is unknown. We found that HDAC6 inhibitors ACY-1215 (1215) and ACY-738 (738) inhibited the proliferation of multiple patient-derived and mouse glioma cells. While both inhibitors triggered rapid increases in acetylated alpha-tubulin (aaTub) in the cytosol and led to increased frequencies of primary cilia, they unexpectedly reduced the levels of aaTub in the cilia. To test whether the antiproliferative effects of HDAC6 inhibitors are dependent on tumor cell cilia, we generated patient-derived glioma lines devoid of cilia through depletion of ciliogenesis genes ARL13B or KIF3A. At low concentrations, 1215 or 738 did not decrease the proliferation of cilia-depleted cells. Moreover, the differentiation of glioma cells that was induced by HDAC6 inhibition did not occur after the inhibition of cilia formation. These data suggest HDAC6 signaling at primary cilia promotes the proliferation of glioma cells by restricting their ability to differentiate. Surprisingly, overexpressing HDAC6 did not reduce cilia length or the frequency of ciliated glioma cells, suggesting other factors are required to control HDAC6-mediated cilia disassembly in glioma cells. Collectively, our findings suggest that HDAC6 promotes the proliferation of glioma cells through primary cilia.

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P11.06 Non epigenetic effect of vorinostat in glioblastoma cells

Neuro-Oncology ◽

10.1093/neuonc/noz126.152 ◽

2019 ◽

Vol 21 (Supplement_3) ◽

pp. iii43-iii43

Author(s):

T Perez ◽

R Berges ◽

H Maccario ◽

D Braguer ◽

S Honoré

Keyword(s):

Side Effects ◽

Low Dose ◽

Therapeutic Option ◽

Hdac Inhibitors ◽

Time Lapse ◽

Glioblastoma Cells ◽

Tumor Cell Death ◽

Alpha Tubulin ◽

Cellular Models ◽

Microtubular Cytoskeleton

Abstract BACKGROUND Glioblastoma multiform (GBM) is the most frequent primitive brain tumor. GBM has a high recurrence and mortality. Histone deacetylase (HDAC) inhibitors have evoked great interest because they are able to change transcriptomic profiles to promote tumor cell death but also show undesirable side effects due to the lack of selectivity.We show new properties of low dose vorinostat, which inhibits cytoplasmic HDAC6 and display interesting non-epigenetics effects, especially on the microtubular cytoskeleton. MATERIAL AND METHODS We used murine (GL261) and human (U87 and GBM6 stem cells) cellular models. The cell proliferation was assessed by MTT tests, the migration by the 24 hours Transwell technic and by wound/healing tests. The expression levels of proteins of interest were assessed by Western Blot. Microtubules dynamics were assessed by time-lapse videomicroscopy. The synergy between drugs was tested by Loewe model. RESULTS Vorinostat inhibited the proliferation and the migration of the three cell lines mentioned above at level below the EC50.Vorinostat induces tubulin acetylation and alpha-tubulin c-terminal detyrosination that signed microtubular stabilization and these effects are independent of histone acetylation (HADC3). Interestingly, vorinostat decreases EB1 expression (a bad prognostic factor in GBM) and decreases microtubule dynamics. Moreover, vorinostat decreases neural markers such as GFAP, beta3-tubulin and CNPase and increases mural markers expression such as SMA/EGFR and PDGFR. Finally, it showed a synergy combined with erlotinib. CONCLUSION Low dose vorinostat, which do not affect histone désacetylase, has antitumor effect on glioblastoma cells by a new mechanism involving microtubule cytoskeleton. Interestingly, combination of low doses vorinostatand erlotinibshowed a strong synergy. Low dose, vorinostat could therefore represent an interesting therapeutic option and fewer side effects and that could be used to increased GBM patient sensitivity to erlotinib.

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Loss of Deacetylation Enzymes Hdac6 and Sirt2 Promotes Acetylation of Cytoplasmic Tubulin, but Suppresses Axonemal Acetylation in Zebrafish Cilia

Frontiers in Cell and Developmental Biology ◽

10.3389/fcell.2021.676214 ◽

2021 ◽

Vol 9 ◽

Author(s):

Paweł K. Łysyganicz ◽

Niedharsan Pooranachandran ◽

Xinming Liu ◽

Kathryn I. Adamson ◽

Katarzyna Zielonka ◽

...

Keyword(s):

Histone Deacetylases ◽

Primary Cilia ◽

Extracellular Fluid ◽

Histone Acetyltransferases ◽

Compensatory Mechanism ◽

Post Translational Modification ◽

Zebrafish Model ◽

Alpha Tubulin ◽

Tubulin Acetylation ◽

Sensory Cilia

Cilia are evolutionarily highly conserved organelles with important functions in many organs. The extracellular component of the cilium protruding from the plasma membrane comprises an axoneme composed of microtubule doublets, arranged in a 9 + 0 conformation in primary cilia or 9 + 2 in motile cilia. These microtubules facilitate transport of intraflagellar cargoes along the axoneme. They also provide structural stability to the cilium, which may play an important role in sensory cilia, where signals are received from the movement of extracellular fluid. Post-translational modification of microtubules in cilia is a well-studied phenomenon, and acetylation on lysine 40 (K40) of alpha tubulin is prominent in cilia. It is believed that this modification contributes to the stabilization of cilia. Two classes of enzymes, histone acetyltransferases and histone deacetylases, mediate regulation of tubulin acetylation. Here we use a genetic approach, immunocytochemistry and behavioral tests to investigate the function of tubulin deacetylases in cilia in a zebrafish model. By mutating three histone deacetylase genes (Sirt2, Hdac6, and Hdac10), we identify an unforeseen role for Hdac6 and Sirt2 in cilia. As expected, mutation of these genes leads to increased acetylation of cytoplasmic tubulin, however, surprisingly it caused decreased tubulin acetylation in cilia in the developing eye, ear, brain and kidney. Cilia in the ear and eye showed elevated levels of mono-glycylated tubulin suggesting a compensatory mechanism. These changes did not affect the length or morphology of cilia, however, functional defects in balance was observed, suggesting that the level of tubulin acetylation may affect function of the cilium.

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Microtubules containing acetylated alpha-tubulin in mammalian cells in culture.

The Journal of Cell Biology ◽

10.1083/jcb.104.2.289 ◽

1987 ◽

Vol 104 (2) ◽

pp. 289-302 ◽

Cited By ~ 541

Author(s):

G Piperno ◽

M LeDizet ◽

X J Chang

Keyword(s):

Monoclonal Antibody ◽

Hela Cells ◽

Mammalian Cells ◽

Primary Cilia ◽

Mitotic Spindles ◽

Alpha Tubulin ◽

Cells In Culture ◽

Antimitotic Drugs ◽

Selective Stabilization ◽

Cytoplasmic Microtubules

The subcellular distribution of microtubules containing acetylated alpha-tubulin in mammalian cells in culture was analyzed with 6-11B-1, a monoclonal antibody specific for acetylated alpha-tubulin. Cultures of 3T3, HeLa, and PtK2 cells were grown on coverslips and observed by immunofluorescence microscopy after double-staining by 6-11B-1 and B-5-1-2, a monoclonal antibody specific for all alpha-tubulins. The antibody 6-11B-1 binds to primary cilia, centrioles, mitotic spindles, midbodies, and to subsets of cytoplasmic microtubules in 3T3 and HeLa cells, but not in PtK2 cells. These observations confirm that the acetylation of alpha-tubulin is a modification occurring in different microtubule structures and in a variety of eukaryotic cells. Some features of the acetylation of cytoplasmic microtubules of mammalian cells are also described here. First, acetylated alpha-tubulin is present in microtubules that, under depolymerizing conditions, are more stable than the majority of cytoplasmic microtubules. In addition to the specific microtubule frameworks already mentioned, cytoplasmic microtubules resistant to nocodazole or colchicine, but not cold-resistant microtubules, contain more acetylated alpha-tubulin than the rest of cellular microtubules. Second, the alpha-tubulin in all cytoplasmic microtubules of 3T3 and HeLa cells becomes acetylated in the presence of taxol, a drug that stabilizes microtubules. Third, acetylation and deacetylation of cytoplasmic microtubules are reversible in cells released from exposure to 0 degrees C or antimitotic drugs. Fourth, the epitope recognized by the antibody 6-11B-1 is not absolutely necessary for cell growth and division. This epitope is absent in PtK2 cells. The acetylation of alpha-tubulin could regulate the presence of microtubules in specific intracellular spaces by selective stabilization.

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