Acetylated Tubulin Research Articles

P-103 Three-dimensional oviductal organoid model to study spermatozoa-oviduct interaction in humans

Abstract Study question Is it possible to establish an in vitro model that recapitulating human oviduct to investigate sperm-oviduct interaction Summary answer Three-dimensional (3D) oviductal organoid model was established from either primary human oviductal tissues or immortalized human OE-E6/67 cell line, based on our protocol. What is known already The sperm reservoir in oviduct is formed to maintain the fertilizing capacity of spermatozoa until ovulation by the binding of sperm to the epithelial lining of the oviduct, which is also significant to select sperm with normal chromatin structure and superior fertilization ability. The regulation and mechanism of sperm reservoir formation in human is still unknown, mainly due to the ethical issue and technical difficulty in obtaining enough human oviduct. We previously demonstrated that sperm fucosyltransferase-5 (sFUT5), a carbohydrate-binding protein on spermatozoa, is responsible for spermatozoa-oviduct interaction in humans. Study design, size, duration Human oviductal epithelial cell line immortalized by HPV 16 E6/E7 open reading frame by retroviral expression system was employed to establish the co-culture model. Fresh human samples (human spermatozoa,N = 30; 1-2 cm primary oviductal tissue,N = 15) were collected in hospital with signed consent. Fluorescent pre-labelled sperm were co-cultured with organoids for 2 hours. At least three replicates were conducted for each assay. Participants/materials, setting, methods Morphological characterization and markers expressions of the organoid were determined by immunohistochemistry and confocal microscopy analysis. The sperm binding capacity of the oviductal organoid was studied by co-culture system. Sperm chromatin structure of the organoid-bound sperm was assessed by flow cytometry-based TUNEL assay. Affinity chromatography followed by nano-liquid chromatography–mass spectrometry was used to identify the sFUT5-binding proteins on ovidutal epithelial cells (OECs) and their expressions on OECs were validated by immunostaining/flow cytometry. Main results and the role of chance The organoid exhibited a hollow structure surrounded by a single layer of OECs. Immunohistochemistry analysis revealed the high expressions of epithelial marker E-cadherin (basolateral membrane), secretory marker pair-box 8 (PAX8) and proliferative marker Ki67. Expression of ciliated cell marker, acetylated tubulin (AcTUB) can also be detected. The modification of culture environment successfully reversed the OEC organoids polarity as demonstrated by immunostaining using antibodies against ZO-1 (apical marker) and E-cadherin (basolateral marker), making the organoid accessible during the co-culture. These apical-out OEC organoids have significantly higher sperm binding capacities than their basal-out counterparts. Furthermore, OEC organoid-bound spermatozoa possessed more intact DNA than the unbound spermatozoa. The results indicate the relevance of using OEC organoid-spermatozoa co-culture model as a tool to assess spermatozoa-oviduct interaction in humans. By using immuno-affinity chromatography and mass spectrometry analysis, cell adhesion molecule 4 (CADM4) was then identified as a potential sFUT5-interacting protein on OECs. This result was further supported by flow cytometry and immunofluorescent staining. 3-D oviductal organoid model demonstrates profound potential for in vitro investigation of human oviduct physiology. Limitations, reasons for caution Our 3D model may not fully represent the entire cellular complexity and tubular architecture of the oviductal epithelium. Further investigation on the sperm morphology and DNA integrity and analysis on organoid transcriptome will enhance our understanding on oviduct in human reproduction. Wider implications of the findings The results from co-culture may give a clue to future study on whether oviductal organoid can be applied clinically for functional sperm selection in in vitro fertilization. Our apical-out organoid model may also be applied to study tubal ectopic pregnency. Trial registration number not applicable

Abstract 965: 5-Aza-dC treatment impacts the microtubule network to decrease metastatic potential through reduced cell clustering and attachment

Abstract Melanoma cell adhesion molecule (MCAM) is a protein located on the cell surface that was first discovered in advanced primary tumors and metastatic lesions. Overexpression of MCAM is associated with triple-negative breast cancer, decreased patient survival, and induces vimentin and slug indicative of an epithelial to mesenchymal transition (EMT). Additionally, breast cancer cell lines overexpressing MCAM show an increase in migration, invasion, and tumorigenesis. Given that overexpressed MCAM causes increased migration and invasion in multiple cancer types, MCAM could be one of the major contributors to recent studies showing increased metastatic phenotypes with certain cancer treatments. Decitabine (5-Aza-dC) is a chemotherapeutic used for acute myeloid leukemias with clinical trials underway for breast cancer use. 5-Aza-dC results in the depletion of DNA methyltransferase and acts as a demethylating agent. In 2019, 5-Aza-dC was reported to increase mRNA and protein levels of MCAM in various breast cancer cell lines, followed by an increase in EMT markers. Studies suggest that 5-Aza-dC treatment in chemo-sensitive ovarian cancer cell lines results in increased EMT markers as well as increased invasion and migration. Microtubules are a very common target of multiple cancer therapies. However, the impact of 5-Aza-dC and MCAM on the microtubule network and non-adherent cells has not been well studied. We set out to explore changes in microtentacle (McTN), cell attachment and clustering, and microtubule modifications due to 5-Aza-dC treatment and MCAM expression. Treatment of MCF10A, MCF10A variants, and MCF7 breast cancer cell lines with 5-Aza-dC showed consistent increases in the expression of MCAM. When exploring the effects of 5-Aza-dC treatment on the cytoskeleton, specifically microtubules, it was found that there is a significant increase in acetyl tubulin and detyrosinated tubulin, two common microtubule modifications correlated with an increase of McTNs. However, there was not a significant increase in the number of cells with McTNs in treated cells. Since McTNs can aid in attachment and cell clustering, attachment assays and clustering assays were performed after 5-Aza-dC treatment. These experiments showed that 5-Aza-dC decreased the ability of all cell lines to attach and impeded these cells' ability to form clusters. While there are emerging concerns that Decitabine can increase metastatic phenotypes of migration and invasion in adherent cells, our results indicate that within a non-adherent environment, Decitabine reduces the metastatic phenotypes of clustering and reattachment. Ongoing experiments focus on testing the impact of stable genetic alterations in these cell lines to examine the specific impact of MCAM overexpression on the microtubule network and metastatic potential. Citation Format: David Annis, Keyata Thompson, Julia Ju, Makenzy Mull, Trevor Mathias, Michele Vitolo, Stuart Martin. 5-Aza-dC treatment impacts the microtubule network to decrease metastatic potential through reduced cell clustering and attachment [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 965.

Acetylation/deacetylation and microtubule associated proteins influence flagellar axonemal stability and sperm motility.

PTMs and microtubule-associated proteins (MAPs) are known to regulate microtubule dynamicity in somatic cells. Reported literature on modulation of α-tubulin acetyl transferase (αTAT1) and histone deacetylase 6 (HDAC6) in animal models and cell lines illustrate disparity in correlating tubulin acetylation status with stability of MT. Our earlier studies showed reduced acetyl tubulin in sperm of asthenozoospermic individuals. Our studies on rat sperm showed that on inhibition of HDAC6 activity, although tubulin acetylation increased, sperm motility was reduced. Studies were therefore undertaken to investigate the influence of tubulin acetylation/deacetylation on MT dynamicity in sperm flagella using rat and human sperm. Our data on rat sperm revealed that HDAC6 specific inhibitor Tubastatin A (T) inhibited sperm motility and neutralized the depolymerizing and motility debilitating effect of Nocodazole. The effect on polymerization was further confirmed in vitro using pure MT and recHDAC6. Also polymerized axoneme was less in sperm of asthenozoosperm compared to normozoosperm. Deacetylase activity was reduced in sperm lysates and axonemes exposed to T and N+T but not in axonemes of sperm treated similarly suggesting that HDAC6 is associated with sperm axonemes or MT. Deacetylase activity was less in asthenozoosperm. Intriguingly, the expression of MDP3 physiologically known to bind to HDAC6 and inhibit its deacetylase activity remained unchanged. However, expression of acetyl α-tubulin, HDAC6 and microtubule stabilizing protein SAXO1 was less in asthenozoosperm. These observations suggest that MAPs and threshold levels of MT acetylation/deacetylation are important for MT dynamicity in sperm and may play a role in regulating sperm motility.

Crafting Carbazole-Based Vorinostat and Tubastatin-A-like Histone Deacetylase (HDAC) Inhibitors with Potent in Vitro and in Vivo Neuroactive Functions.

Small-molecule inhibitors of HDACs (HDACi) induce hyperacetylation of histone and nonhistone proteins and have emerged as potential therapeutic agents in most animal models tested. The established HDACi vorinostat and tubastatin-A alleviate neurodegenerative and behavioral conditions in animal models of neuropsychiatric disorders restoring the neurotrophic milieu. In spite of the neuroactive pharmacological role of HDACi (vorinostat and tubastatin-A), they are limited by efficacy and toxicity. Considering these limitations and concern, we have designed novel compounds 3–11 as potential HDACi based on the strategic crafting of the key pharmacophoric elements of vorinostat and tubastatin-A into architecting a single molecule. The molecules 3–11 were synthesized through a multistep reaction sequence starting from carbazole and were fully characterized by NMR and mass spectral analysis. The novel molecules 3–11 showed remarkable pan HDAC inhibition and the potential to increase the levels of acetyl H3 and acetyl tubulin. In addition, few novel HDAC inhibitors 4–8, 10, and 11 exhibited significant neurite outgrowth-promoting activity with no observable cytotoxic effects, and interestingly, compound 5 has shown comparably more neurite growth than the parent compounds vorinostat and tubastatin-A. Also, compound 5 was evaluated for possible mood-elevating effects in a chronic unpredictable stress model of Zebrafish. It showed potent anxiolytic and antidepressant-like effects in the novel tank test and social interaction test, respectively. Furthermore, the potent in vitro and in vivo neuroactive compound 5 has shown selectivity for class II over class I HDACs. Our results suggest that the novel carbazole-based HDAC inhibitors, crafted with vorinostat and tubastatin-A pharmacophoric moieties, have potent neurite outgrowth activity and potential to be developed as therapeutics to treat depression and related psychiatric disorders.

Expression of acetylated tubulin in the postnatal developing mouse cochlea.

Microtubules are an essential component of the cytoskeleton of a eukaryotic cell. The post-translational tubulin modifications play an important role in regulating microtubule properties, acetylation tubulin is one of the major post-translational modifications of microtubules. Acetylation tubulin has also been shown to be expressed in the cochlea. However, the detailed expression profiles of acetylation tubulin protein during development have not yet been investigated in the postnatal mammalian cochlea. Here, we first examined the spatio-temporal expression of acetylated tubulin in the mouse cochlea during postnatal development. At postnatal day 1 (P1), acetylated tubulin was localized primarily to the auditory nerve inside the cochlea and their synaptic contacts with the inner and outer hair cells (IHCs and OHCs). In the organ of Corti, acetylated tubulin occurred first at the apex of pillar cells. At P5, acetylated tubulin first appeared in the phalangeal processes of Deiters’ cells. At P8, staining was maintained in the phalangeal processes of Deiters’ cells and neural elements. At P10, labeling in Deiters’ cells extended from the apices of OHCs to the basilar membrane, acetylated tubulin was expressed throughout the cytoplasm of inner and outer pillar cells. At P12, acetylated tubulin displayed prominent and homogeneous labeling along the full length of the pillar cells. Linear labeling was present mainly in the Deiters’ cell bodies underlying OHCs. Between P14 and P17, acetylated tubulin was strongly expressed in inner and outer pillar cells and Deiters’ cells in a similar pattern as observed in the adult, and labeling in these cells were arranged in bundles. In addition, acetylated tubulin was expressed in stria vascularis, root cell bodies, and a small number of fibrocytes of the spiral ligament until the adult. In the adult mouse cochlea, immunostaining continued to predominate in Deiters’ cells and pillar cells. In Deiters’ cells, immunolabeling formed cups securing OHCs basal portions, and continued presence of acetylated tubulin-labeled nerve terminals below IHCs was shown. Our results presented here underscored the essential role played by acetylated tubulin in postnatal cochlear development, auditory neurotransmission and cochlear mechanics.

Abstract 4976: Ricolinostat, a selective HDAC6 inhibitor with immunomodulatory properties, has significant antimelanoma activity in vitro and in vivo

Abstract Melanoma is a less common but the most aggressive type of skin cancer and the rates of melanoma have been rising for the last 30 years. Immunotherapy may represent a new treatment paradigm to benefit melanoma patients. Previous studies have identified HDAC6 playing a central role in the regulating melanoma immunogenicity. We have shown that genetic knockdown (KD) of HDAC6 in murine and human melanoma cells resulted in an increased the expression of MHCI and costimulatory molecules as well as melanoma associated antigens. In vivo studies demonstrated that the growth of melanoma cells lacking HDAC6 was significantly delayed as compared to wild-type cells. This growth was further delayed when HDAC6KD melanoma bearing mice were treated with anti-PD-1 antibodies. Recently, we have shown that in vitro treatment of murine melanoma cells with Ricolinostat decreases PD-L1 expression and increases the expression of acetyl tubulin. Surprisingly, in vivo treatment of B16 melanoma bearing animals with increasing concentrations of ricolinostat resulted in a dose-dependent inhibition of melanoma growth (p<0.05). No toxicities were observed at the doses studied; however, the antitumor effect exerted by ricolinostat was not observed when melanoma bearing SCID mice were treated with this compound, indicating that an intact host immune system is required for the observed antitumor activity. In lieu of these previously unknown immunomodulatory properties of ricolinostat, we next assessed the effects of this compound upon T-cells and whether ricolinostat could augment the efficacy of checkpoint blockade in vivo. Murine T-cells were activated with anti-CD3 plus anti-CD28 in the presence or absence of ricolinostat. Then, those T-cells were adoptively transferred into B16 melanoma bearing mice. T-cell subpopulations from the lymph nodes were analyzed ex vivo. We show here that treatment of murine T-cells with ricolinostat resulted in a significant increase in central memory T-cells endowed with a strong anti-melanoma activity in vivo as compared to control group (p<0.05). Finally, the addition of ricolinostat treatment to either anti-CTLA4 or anti-PD1 treatment was associated with an enhanced inhibition of melanoma tumor growth. In summary, our results have identified HDAC6 as a novel target for melanoma immunotherapy and point out ricolinostat as an attractive agent to add to the immuno-oncology armamentarium. Citation Format: Fengdong Cheng, Sodre Andressa, Jie Chen, Alejandro Villagra, David Woods, Jeffrey Weber, Steven Quayle, Jones Simon, Eduardo Sotomayor. Ricolinostat, a selective HDAC6 inhibitor with immunomodulatory properties, has significant antimelanoma activity in vitro and in vivo [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 4976. doi:10.1158/1538-7445.AM2017-4976

From the Cover: ImpairedProliferation and Differentiation of the Conducting Airway Epithelium Associated With Bronchiolitis Obliterans After Sulfur Mustard Inhalation Injury in Rats.

Adult male rats were exposed to a sublethal dose of SM inhalation (1.0-1.2 mg/kg) for 50 min. Histological sections of the bronchiolar epithelium were analyzed for changes using hematoxylin and eosin, trichrome, and immunofluorescent staining for acetylated tubulin (AT) and club cell secretory protein (CCSP). CCSP in bronchoalveolar lavage fluid was assessed using western blot. A bromodeoxyuridine (BRDU) assay was used to assess for epithelial proliferation, and real-time PCR measured changes in Notch mRNA expression. SM caused significant proximal bronchiolar epithelial injury with epithelial denudation, loss of acetylated tubulin and CCSP staining, and reduced bronchoalveolar lavage fluid CCSP levels. bromodeoxyuridine (BRDU) + staining of proximal bronchiolar epithelial cells was not increased, but staining was increased in the distal bronchiolar epithelium. One month after injury, the proximal bronchiolar epithelium was not fully repaired. Significant collagen deposition surrounded proximal bronchioles with luminal obstruction, consistent with bronchiolitis obliterans. These changes corresponded with a downregulation of Notch1, Notch3, and Hes1 mRNA expressions. This study demonstrates that SM exposure resulted in severe proximal airway epithelial injury, persistent morphological changes, impaired epithelial proliferation and, ultimately, bronchiolitis obliterans. These changes occurred at the same time that the Notch signaling genes were downregulated. Thus, the lung epithelium and the Notch signaling pathway may be worthy targets for the prevention of chronic airway remodeling after SM inhalation injury.

Evidence of Two Ciliated Epithelial Cell Subsets in Mouse Airways

Patients with asthma-associated airway epithelial damage exhale increased levels of nitric oxide (NO). However, the distribution of endothelial NO synthase (eNOS) in mouse airways remains to be controversial. In the present study, mouse lung sections were stained using antibodies against secretoglobin 1A member [1] (Scgb1a1), acetylated tubulin (ACT), and eNOS. We found that club cells in the mouse airways are immunoreactive toward eNOS. In addition, to the best of our knowledge, for the first time, two subsets of ciliated cells that differ in their expression of eNOS were observed to reside in the mouse airways. Both subsets of ciliated cells survived naphthalene-induced lung injury. These data will help clarify a controversial issue of whether ciliated cells contribute to epithelial maintenance in the airways.

Acetylated Tubulin (AT) as a Prognostic Marker in Squamous Cell Carcinoma of the Head and Neck

Acetylated tubulin (AT) expression has been proposed as a marker for sensitivity to taxane chemotherapy. We wanted to explore AT as a prognostic marker in squamous cell carcinoma of the head and neck (SCCHN). We assessed AT expression in archival tissue from our institutional tissue bank of primary SCCHN specimens. We also examined AT expression on pre-therapy tissues of patients with SCCHN receiving induction chemotherapy with docetaxel, cisplatin and 5FU (TPF IC). AT expression was assessed on archival cases of SCCHN with (N = 63) and without (N = 82) locoregional lymph node metastases (LNM). The predominant tumor site was oral cavity (52 %). Immunohistochemistry staining was based on staining intensity and percentage of tumor cells stained to create a weighted index (WI). A total of nine patients who received TPF IC were evaluable for response by RECIST and also had pre-therapy tissues available. A significant independent correlation between AT and tumor grade (p = 0.001) and primary location (p = 0.008) was noted. There was a trend of higher AT in patients with presence of LNM (p = 0.052) and a trend in improved OS for patients with an AT WI below the median compared to those above the median for patients with no LNM (p = 0.054). For patients treated with induction TPF, we observed an inverse correlation between AT expression and response to TPF IC (p = 0.0071). AT expression is correlated with tumor grade and primary site. There was an observed trend correlating AT with presence nodal metastases. The observed inverse correlation with response to taxane based chemotherapy needs validation in a larger sample size.

Abstract 3744: Acetylated tubulin (AT) levels predicts response to TPF chemotherapy in locally advanced head and neck squamous cell carcinoma (HNSCC)

Abstract Docetaxel acts by binding to β-tubulin, stabilizing microtubules, and causing cell cycle arrest in the G2/M phase of mitosis. Previous reports by our group suggest the absence of acetylated tubulin in tumor samples is a predictor for disease control in patients treated on a phase I trial with Docetaxel and Lonafarnib[1]. In this study, we hypothesize that patients with HNSCC with low levels of microtubule acetylation (i.e. highly dynamic microtubules) as measured by immunohistochemistry (IHC) would respond better to induction therapy with TPF (Docetaxel, Cisplatin and 5FU), and hence the level of acetylated tubulin (AT) would be a predictor of response to TPF. Method: Between July 5th 2006 and September 29th2009, 14 patients with HNSCC were enrolled in a Phase II clinical trial of induction chemotherapy with 3 cycles of TPF followed by local therapy. Patients underwent a biopsy of their primary tumor site prior to initiation of induction TPF. The IHC staining for AT expression was performed on formalin fixed paraffin embedded tissue. The scoring was based on intensity only since tumor cells displayed a homogeneous staining pattern. An AT score of 4 was considered to be high, whereas a stain of 0, 1, 2 or 3 was considered low. Primary tumor sites included oral cavity (OC; n=6), oropharynx (OP; n=5), larynx (L; n=1), sinus (S; n=1), and unknown primary (UP; n=1). All patients had stage IVa or IVb at presentation and had a performance status of 0-2 (Eastern Cooperative Oncology Group ECOG scale). All had at least one uni- or bi-dimensional measurable lesion on imaging. The study closed prematurely due to slow accrual and increased observed complications. A total of 9 patients were evaluated: OC/4, OP/5. Results: After 3 cycles of TPF induction chemotherapy, complete remission (CR) was achieved in 4/9(44.5%), partial remission (PR) in 4/9(44.5%), and progressive disease (PD) in 1/9(11%) of patients. There was a significant correlation between AT staining intensity and response to TPF (p=0.0476). Patients with complete response (CR) or good partial response (GPR) (>60% reduction in tumor measurement) had a significantly lower staining intensity. No correlation was observed between staining intensity and progression free survival (PFS) or overall survival (OS) perhaps due to the small sample size. Conclusion: AT expression seems to correlate with response to TPF chemotherapy in HNSCC. Predicting response to TPF may prove to be useful in customizing care for patients with HNSCC. We are planning a larger scale retrospective analysis of the value of AT expression in predicting outcome for patients with HNSCC. 1. Kauh J, et al., Defining the Interaction of Docetaxel and Lonafarnib in Patients with Advanced Malignancies. AACR meeting, 2008, LB-67, 2008. 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 3744.

Sensory Setae on the Major Chelae of Male Crayfish, Orconectes Rusticus (Decapoda: Astacidae) – Impact of Reproductive State on Function and Distribution

Abstract The major chelae of reproductive male crayfish contain both smooth and plumose setae and are used for the perception of female odors. A comparative and morphological analysis of setae found on the major chelae of reproductive (form I) and non-reproductive (form II) male crayfish, Orconectes rusticus was performed in order to elucidate the distribution and function of smooth and plumose setae. In particular, the distribution of setae between reproductive forms was quantified as well as putative sensory functions, based on morphological characteristics, for both smooth and plumose setae. To accomplish these goals, scanning electron microscopy, a porosity assay, anterograde labeling, and acetylated tubulin (AT) immunocytochemistry were used. We found that form I crayfish have significantly more pockets of sensory setae and individual smooth setae on their major chelae when compared to form II males. Scanning electron microscopy revealed a terminal apical pore-like structure in smooth setae that was ...