Trends Cell Biol Research Articles

Notch-dependent Abl signaling regulates cell motility during ommatidial rotation in Drosophila.

A collective cell motility event that occurs during Drosophila eye development, ommatidial rotation (OR), serves as a paradigm for signaling-pathway-regulated directed movement of cell clusters. OR is instructed by the EGFR and Notch pathways and Frizzled/planar cell polarity (Fz/PCP) signaling, all of which are associated with photoreceptor R3 and R4 specification. Here, we show that Abl kinase negatively regulates OR through its activity in the R3/R4 pair. Abl is localized to apical junctional regions in R4, but not in R3, during OR, and this apical localization requires Notch signaling. We demonstrate that Abl and Notch interact genetically during OR, and Abl co-immunoprecipitates in complexes with Notch in eye discs. Perturbations of Abl interfere with adherens junctional organization of ommatidial preclusters, which mediate the OR process. Together, our data suggest that Abl kinase acts directly downstream of Notch in R4 to fine-tune OR via its effect on adherens junctions.

Coordinate control of basal epithelial cell fate and stem cell maintenance by core EMT transcription factor Zeb1.

Maintenance of undifferentiated, long-lived, and often quiescent stem cells in the basal compartment is important for homeostasis and regeneration of multiple epithelial tissues, but the molecular mechanisms that coordinately control basal cell fate and stem cell quiescence are elusive. Here, we report an epithelium-intrinsic requirement for Zeb1, a core transcriptional inducer of epithelial-to-mesenchymal transition, for mammary epithelial ductal side branching and for basal cell regenerative capacity. Our findings uncover an evolutionarily conserved role of Zeb1 in promoting basal cell fate over luminal differentiation. We show that Zeb1 loss results in increased basal cell proliferation at the expense of quiescence and self-renewal. Moreover, Zeb1 cooperates with YAP to activate Axin2 expression, and inhibition of Wnt signaling partially restores stem cell function to Zeb1-deficient basal cells. Thus, Zeb1 is a transcriptional regulator that maintains both basal cell fate and stem cell quiescence, and it functions in part through suppressing Wnt signaling.

Abstract P199: Tenfibgen nanoencapsulated RNAi feCK2 inhibits protein kinase CK2 and induces apoptosis in feline oral squamous cell carcinomas in vivo

Abstract In an initial safety evaluation in domestic cats suffering from oral squamous cell carcinomas (FOSCC), nine cat subjects were treated IV with 5-6 doses given twice weekly of s50 RNAi feCK2 at either 2 or 20 ug/kg (Cannon et. al., Hu Gene Ther Clin Devel 28(2):80-6, 2017). Treatment was generally well tolerated. In one case, a clear drug-related Grade 3 side effect was rapid tumor death (tumor lysis) resulting in an open wound. This issue has been addressed successfully in separate mice studies with longer, lower-dose SQ regimens. Protein kinase CK2, as a promising target in oncology, plays vital roles in cell growth, proliferation, and suppression of apoptosis (Ahmed et al., Trends Cell Biol 12: 226-30, 2002). Anti-feCK2aa’ is a tumor-targeted 20 nm crystalline capsule bearing single-stranded RNAi oligos against both CK2 catalytic kinase subunits with a ligand-coated shell derived from Tenascin-C (tenfibgen). Tenascin-C is upregulated in multiple solid tumors and is present in tumors throughout their lifecycle. The capsule’s ultrasmall size enables efficient raft-mediated delivery of oligos to the perinuclear space of the target cell, and the ability to reach metastases without reliance on EPR (Unger et. al, Mol Cancer Ther 13:2018-29, 2014). Initial mechanistic work focused on changes in clinically scored IHC of the CK2a subunit in study tumors. In an effort to develop a future working hypothesis, remaining paraffin-embedded tissues were retrospectively examined for both kinase subunits of CK2 as a mixture (CK2aa’), cleaved PARP (CP, apoptosis marker) and Ago2 (critical enzyme required for RNAi drug activity). Previous work in murine models showed variation of Ago2 levels in primary human and xenograft tumors. Further, model xenograft and syngeneic tumors responded with increased Ago2 levels and drug activity upon 10 mg/ml IV priming followed by low-dose SQ treatment (Cancer Res 2016;76(14 Suppl):3746). Using confocal fluorescence microscopy, we observed that decreased post-treatment CK2aa’ and increased CP corresponded positively with clinical tumor responses while non-responders showed an inverse relationship. Examination of arrayed human HNC samples also showed a variation in Ago2 levels but premalignant oral lesions from a human chemoprevention trial did not. These observations suggest a possible future path for feline patient therapy of advanced lesions using Ago2 stratification, potential IV priming and more convenient, longer-term SQ dosing administered by feline caregivers. A path for human therapy may lie through chemoprevention with much to be learned from feline studies. Funding: University of Minnesota Clinical and Translational Sciences Award (1UL1 RR033183-01), NIH SBIR Contract HHSN26120 1300030C, N01-CN-15000, Mod#5, V.A. Merit Review research funds (BX00173 and BX003282) and NCI Award CA150182. Citation Format: Frank G. Ondrey, Gretchen M. Unger, Claire M. Cannon, Janeen H. Trembley, BT Kren, Jaime F. Modiano, Khalil Ahmed. Tenfibgen nanoencapsulated RNAi feCK2 inhibits protein kinase CK2 and induces apoptosis in feline oral squamous cell carcinomas in vivo [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr P199.

Bacterial minicells to the rescue: cyto-Immunotherapy for the treatment of late stage cancers with minimal to no toxicity.

Bacterial minicells to the rescue: cyto-Immunotherapy for the treatment of late stage cancers with minimal to no toxicity.

Cancer associated fibroblasts: the major player in pancreatic cancer

Tumor cells are not alone in the tumor mass; in fact they are surrounded by a complex and active microenvironment, composed by fibroblasts and their extracellular matrix (ECM), immune cells, nerves, blood vessels, and secreted factors. It is now well recognized that the microenvironment plays an important role in tumor development and thus it is imperative for the comprehensive understanding of the interplay between cancer and its microenviroment for the development of better preventative and therapeutic strategies. Pancreatic ductal adenocarcinoma (PDAC) is a devastating disease with a 5 year survival rate of only 10% after diagnosis[1]. One of the main reasons for this outcome is the poor understanding of the unique microenvironment of PDAC, where up to 90% of the tumor mass can be composed by the stroma, with most of it being the expansion of activated fibroblasts and their ECM[2]. Even though CAFs represent an important component of PDAC (and other types of cancers), they are still incompletely understood. This can be partially explained by the fact that there are no specific markers to discriminate CAFs, and researchers need to rely on negative selections, absence of mutations that characterize the transformed epithelial cells plus the presence of mesenchymal markers, all together with the assessment of fibroblastic function2. There are still controversies about the pro- and anti-tumor effects of CAFs and their origin. Recently, a consensus was published2, where authors suggest that CAFs are mostly originated by the local activation and proliferation of resident fibroblasts, stimulated by tissue injury, reactive oxidative species, growth factors and more. These CAFs are characterized by their plasticity, as they can interchange between functions according to the signals of the environment. Many are the functions attributed to these cells; CAFs produce a very dense ECM, which can lead to the collapse of blood vessels, thus affecting nutrient supply and the delivery of therapies to this environment[3]. Moreover, CAFs can be immunusuppressive, producing a millieu of cytokines and chemokines that can turn off anti-tumor immune cells and recruit pro-tumor ones[4]. Interestingly, CAFs can also contribute to the metabolic signature of the environment, as they produce and modify a large range of metabolites, often supporting cancer cell survival[5]. Recently, our research group uncovered the pro-tumor roles of the synaptic protein Netrin G1 in CAFs[6]. The expression of Netrin G1 in CAFs from PDAC patients inversely correlated with overall survival. Moreover, the loss of Netrin G1 in CAFs led to decreased production of immunosuppressive factors, allowing NK cells to kill PDAC cells in vitro. Netrin G1 expression was also important for the production of metabolites (mainly glutamine and glutamate) by CAFs and for the support of nutrient deprived PDAC cells. It is important to mention that the Netrin G1 related studies were perfomed using 3D co-cultures, and that its expression can only be detected in CAFs growing in their 3D environment, further reinforcing the need for better strategies to study and understand the tumor microenvironment in PDAC. Therefore, CAFs can be seen as the major intermediaries of PDAC microenvironment and to target these cells in an attempt to normalize them rather than eliminate them, might be an effective strategy for PDAC therapy.

Autophagy in Drosophila and Zebrafish.

Autophagy is a highly conserved cellular process that delivers cellular contents to the lysosome for degradation. It not only serves as a bulk degradation system for various cytoplasmic components but also functions selectively to clear damaged organelles, aggregated proteins, and invading pathogens (Feng et al., Cell Res 24:24-41, 2014; Galluzzi et al., EMBO J 36:1811-36, 2017; Klionsky et al., Autophagy 12:1-222, 2016). The malfunction of autophagy leads to multiple developmental defects and diseases (Mizushima et al., Nature 451:1069-75, 2008). Drosophila and zebrafish are higher metazoan model systems with sophisticated genetic tools readily available, which make it possible to dissect the autophagic processes and to understand the physiological functions of autophagy (Lorincz et al., Cells 6:22, 2017a; Mathai et al., Cells 6:21, 2017; Zhang and Baehrecke, Trends Cell Biol 25:376-87, 2015). In this chapter, we will discuss recent progress that has been made in the autophagic field by using these animal models. We will focus on the protein machineries required for autophagosome formation and maturation as well as the physiological roles of autophagy in both Drosophila and zebrafish.

Consideration of Pannexin 1 channels in COVID-19 pathology and treatment.

Pannexin1 (PANX1) is a ubiquitously expressed, channel-forming protein found in a number of tissues thoughout the body (e.g. lung, vasculature, liver, central nervous system, immune system) that is important in many key physiological and immune responses. PANX1 channels passively flux ATP (predominantly), multiple metabolites, and likely other small anions. PANX1 channels regulate inflammation and host responses to several pathogens, including viruses. While there is currently no evidence suggesting novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and PANX1 directly interact, there is an urgent need for therapeutic strategies, especially those targeting the hyper-inflammation and cytokine storm that occurs in severe cases of COVID-19. Here we argue that PANX1, and drugs known to target PANX1 (including the FDA-approved drug probenecid), should be the focus of further investigation in the context of SARS-CoV-2 infection and its associated pathology in COVID-19 patients.

The ER-Localized Transmembrane Protein TMEM39A/SUSR2 Regulates Autophagy by Controlling the Trafficking of the PtdIns(4)P Phosphatase SAC1.

TMEM39A, encoding an ER-localized transmembrane protein, is a susceptibility locus for multiple autoimmune diseases. The molecular function of TMEM39A remains completely unknown. Here we demonstrated that TMEM39A, also called SUSR2, modulates autophagy activity by regulating the spatial distribution and levels of PtdIns(4)P. Depletion of SUSR2 elevates late endosomal/lysosomal PtdIns(4)P levels, facilitating recruitment of the HOPS complex to promote assembly of the SNARE complex for autophagosome maturation. SUSR2 knockdown also increases the degradative capability of lysosomes. Mechanistically, SUSR2 interacts with the ER-localized PtdIns(4)P phosphatase SAC1 and also the COPII SEC23/SEC24 subunits to promote the ER-to-Golgi transport of SAC1. Retention of SAC1 on the ER in SUSR2 knockdown cells increases the level of PtdIns(3)P produced by the VPS34 complex, promoting autophagosome formation. Our study reveals that TMEM39A/SUSR2 acts as an adaptor protein for efficient export of SAC1from the ER and provides insights into the pathogenesis of diseases associated with TMEM39A mutations.

MIM through MOM: the awakening of Bax and Bak pores.

The mechanism whereby mitochondrial DNA (mtDNA) is released into the cytosol and activates the cGAS/STING inflammatory pathway during Bax/Bax‐mediated apoptosis is unknown. In this issue, Riley et al (2018) report that widening of Bax and Bak pores on the mitochondrial outer membrane (MOM) during apoptosis allows the extrusion of the mitochondrial inner membrane (MIM) into the cytosol and its permeabilization to release mtDNA independently of caspases. In this scenario, Bax and Bak emerge as key modulators of the apoptotic immunogenic response.

IQGAP1 Binds The Axl Receptor Tyrosine Kinase And Inhibits Its Signaling

IQGAP1 is a scaffold protein that integrates cell signaling pathways by binding several growth factor receptors and intracellular signaling molecules (Trends Cell Biol 2015; 25:171–184). The Axl tyrosine kinase receptor is important in hematopoiesis, the innate immune response, platelet aggregation, engulfment of apoptotic cells and cell survival (Cold Spring Harb Perspect Biol 2013; 5:a009076). Binding of growth arrest‐specific protein 6 (Gas6) to immunoglobulin‐like (Ig‐like) domains of Axl activates intracellular signaling. However, the mechanism of inactivation of the Axl receptor is poorly understood. Here, we investigate the interaction between IQGAP1 and Axl. Using GST pulldown assays and immunoprecipitation from mammalian cell lysates, we found that IQGAP1 associates with Axl in cells. Analysis with multiple discrete fragments of each protein revealed that the binding is direct and mediated by the IQ domain of IQGAP1 and the Ig‐like domains of Axl. We observed by proximity ligation assays that Axl:IQGAP1 complexes are located in the cytoplasm, and this interaction is reduced by 64% when cells are incubated with Gas6. Consistent with these findings, reducing IQGAP1 levels in cells by CRISPR/Cas9 enhanced the ability of Gas6 to stimulate both Axl phosphorylation and activation of Akt. Moreover, reduction of IQGAP1 in cells promoted Gas6‐stimulated transcription of matrix metalloproteinase (MMP)‐2, MMP‐3, MMP‐9 and urokinase‐type plasminogen activator genes. Finally, we observed by confocal microscopy that IQGAP1 downregulates the heterodimerization of Axl with the epidermal growth factor receptor. In summary, we identify IQGAP1 as a previously undescribed suppressor of Axl. These findings differ from the previously reported functions for IQGAP1 as a scaffold that promotes signaling by growth factor receptors (J Biol Chem 2007; 292:3273–3289; J Biol Chem 2011; 286:29734–29747; J Biol Chem 2011; 286:15010–15021). Our data expand the repertoire of binding partners for both Axl and IQGAP1, and provide additional insight into the regulation of Axl function.Support or Funding InformationThis work was supported by the National Institutes of Health Intramural Research Program.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Back to basics: Refined nuclear reprogramming techniques yield higher-quality stem cells.

Back to basics: Refined nuclear reprogramming techniques yield higher-quality stem cells.

Ron R. Kopito: Unfolding the Secrets of Protein Aggregation

Today the mechanism of protein aggregation is an intensely investigated subject in cell biology. We know that protein aggregation can occur due to a variety of causes including mutations in proteins easily misfolded or disruption of components of protein homeostasis machinery such as chaperones or components of the ubiquitin⿿proteasome system. Moreover, these aggregates are known to increase with age and to be associated with a variety of neurodegenerative disease. But not long ago, our understanding of the fate and function of protein aggregates in cells remained a mystery. Seminal studies revealed that protein aggregates could be sequestered into inclusion bodies called aggresomes through a highly regulated process that relies on dynein-dependent retrograde transport on microtubules. In 2000, Ron R. Kopito from Stanford University described recent insights into the mechanisms of aggregate sequestration and proposed potential benefits of this sequestration for the cell [ 1 Kopito R.R. Aggresomes, inclusion bodies and protein aggregation. Trends Cell Biol. 2000; 10: 524-530 Abstract Full Text Full Text PDF PubMed Scopus (1599) Google Scholar ]. Dr Kopito shares his thoughts on where the field of protein aggregation has come since publication of this review and the outstanding questions that remain.

Anne Ridley: Networking with Rho GTPases.

Members of the Rho family of GTPases act as molecular switches that control a variety of signal transduction pathways. Although discovered in 1985, it was not until the early 1990s that cellular functions were assigned to Rho GTPases. The finding that Rho family proteins regulate actin dynamics revolutionized the field of cell biology, and opened the doors to studying Rho GTPases in a variety of cellular contexts. Over the years, new members of the Rho family of GTPases have been identified as well as their impact on new areas of investigation including cell migration, cancer, microbial infection, developmental biology, and neurobiology. The extensive and ever-unfolding network formed by Rho GTPases has prompted the need for synthesized updates on the subject every few years. Two reviews from Anne Ridley from King's College London have provided the field with a growing appreciation and understanding of how the regulation and function of Rho GTPases have evolved since its discovery [ 1 Ridley A.J. Rho family proteins: coordinating cell responses. Trends Cell Biol. 2001; 11: 471-477 Abstract Full Text Full Text PDF PubMed Scopus (639) Google Scholar , 2 Ridley A.J. Rho GTPases and actin dynamics in membrane protrusions and vesicle trafficking. Trends Cell Biol. 2006; 16: 522-529 Abstract Full Text Full Text PDF PubMed Scopus (875) Google Scholar ]. Dr. Ridley shares her thoughts on the complexity of Rho GTPases as well as her advice on writing the perfect review.

TFG Promotes Organization of Transitional ER and Efficient Collagen Secretion.

SummaryCollagen is the most abundant protein in the animal kingdom. It is of fundamental importance during development for cell differentiation and tissue morphogenesis as well as in pathological processes such as fibrosis and cancer cell migration. However, our understanding of the mechanisms of procollagen secretion remains limited. Here, we show that TFG organizes transitional ER (tER) and ER exit sites (ERESs) into larger structures. Depletion of TFG results in dispersion of tER elements that remain associated with individual ER-Golgi intermediate compartments (ERGICs) as largely functional ERESs. We show that TFG is not required for the transport and packaging of small soluble cargoes but is necessary for the export of procollagen from the ER. Our work therefore suggests a key relationship between the structure and function of ERESs and a central role for TFG in optimizing COPII assembly for procollagen export.

ДОСЛІДЖЕННЯ РАНОЗАГОЮВАЛЬНОЇ ДІЇ МАЗІ З ЕКСТРАКТОМ СОСНИ ЗВИЧАЙНОЇ

<p align="center"><strong>RESEARCH OF </strong><strong>THE WOUND </strong><strong>HEALING</strong><strong> ACTION OF THE</strong><strong> OINTMENT WITH PINE EXTRACT</strong><strong></strong></p><p align="center"><strong><sup>1</sup></strong><strong> </strong><strong>T</strong><strong>. </strong><strong>P</strong><strong>. </strong><strong>Mandzii</strong><strong>, <sup>2</sup> А. </strong><strong>R</strong><strong>. </strong><strong>Grytsyk</strong><strong>, <sup>3</sup> А. О. Kl</strong><strong>y</strong><strong>menko, <sup>4</sup> І. О. Nyektyehayev</strong></p><p align="center"><sup>1,2,3</sup> SHEI "Ivano - Frankivsk national medical university", Ivano – Frankivsk</p><p align="center"><sup>4</sup> SHEI "Lviv national medical university named after Danylo Galyts’kiy",</p><p align="center">Lviv</p><p align="center"> </p><p><strong>Introduction</strong><strong>. </strong>The range of medicines intended for the local conservative treatment of wounds is growing steadily. Currently, there is no universal drug suitable for application in all phases of wound healing process: purulent-necrotic, phases of regeneration (the formation of granulation tissue) and scarring, because each phase of treatment requires a differentiated selection of drugs with different mechanisms of action.</p><p>Worked out literature sources have shown the prospect of pine dosage form development. <em>Pinaceae </em>family consists of 10 genera and around 250 species and is the most common group among coniferous class and among gymnosperms in general. <em>Pinus</em> genus includes the greatest number of species. It contains about 100 species represented by evergreen trees. The most widespread in Ukraine is pine (<em>Pinus s</em><em>y</em><em>lvestris</em> L.).</p><p>In medical practice pine buds (<em>Gemmae Pini</em>, or <em>Turiones pini</em>) and products derived from pine, such as turpentine (<em>Oleum Therebintyinae</em>), essential oil (<em>Oleum Pini sylvestris</em>), rosin<em> </em>(<em>Pix liquida Pini</em>) and activated charcoal<em> </em>(<em>Carbo activata</em>) are used.</p><p><strong>Investigation</strong><strong> </strong><strong>methods</strong><strong>.</strong> Wound healing activity was studied in experiments <em>in vivo</em> on 32 white outbred rats according to the "General ethical principles of animal experimentation" (Ukraine, 2001). Experiment was conducted on animals weighing 170 -192 g, who were on a general diet and kept in the same conditions. To reproduct the model of linear wound rats were introduced into barbamil anesthesia (0,8 ml of 1 % aqueous solution at barbami for 100 mg of body weight). After their hair was cut off, a piece of skin sized 1×1 cm was carved in aseptic conditions (operating area was moistened with 5% alcoholic solution of iodine).</p><p><strong>Results and discussion</strong><strong>.</strong><strong> </strong>The obtained results indicate that the acceleration of wound healing process was observed under the application of the ointment with extract of pine (reduction of wound area to 50,10 ± 3,34 and 2,13 ± 0,52 mm<sup>2</sup> on the 7<sup>th</sup> and 14<sup>th</sup> day respectively). Thus, ointment with extract of pine as the medicine for the treatment of wounds in the experiment prevails the referent medicine (group 4) and provides wound healing in a shorter time. Under visual observation of group number 2 the change in wound area on the 7<sup>th</sup> and 14<sup>th</sup> day (50,10 and 2,13 mm<sup>2</sup> respectively) was observed.</p><p>The development of wound process was accompanied by changes in protein metabolism and level of middle mass molecules (total protein of animals of the control group and the group number 2 was 45,6 ± 1,17 g/l and 57,4 ± 2,9 g/l respectively, the content of middle mass molecules was 0,29 ± 0,02 standard units and 0,22 ± 0,01 standard units respectively).</p><p class="aCxSpFirst"><strong>Conclusions</strong><strong>. </strong></p><ol><li>The results of conducted researches show that the application of an ointment with pine extract for the treatment of wound process provides wound healing at a shorter time.</li><li>Reparative activity of the ointment with pine extract applied to the wound area shows more pronounced activity in comparison with the referent medicine.</li></ol><p class="aCxSpMiddle"><strong>References</strong><strong></strong></p><p class="aCxSpMiddle">1. Shalymov O. O. Suchasne medykamentozne likuvannya ran / O. O. Shalymov, V. F. Sayenko, B. M. Datsenko // Vidomcha instruktsiya.– K., 2002.– 37 s.</p><p class="aCxSpMiddle">2. Martin P. Inflammatory cells during wound repair: the good, the bad and the ugly / P. Martin, S. J. Leibovich // Trends Cell Biol.– 2005.– V. 15, № 4.– R. 599–607.</p><p class="aCxSpMiddle">3. Velnar T. The wound healing process: an overview of the cellular and molecular mechanisms / T. Velnar, T. Bailey, V. Smrkolj // J. Int. Med. Res.– 2009.– V. 37, № 5.– R. 1528–1542.</p><p class="aCxSpMiddle">4. Zast·souvannya roslyn klasu khvoyni u medytsyni. Rodyna Sosnovi (Ohlyad literatury) – T. M. Kozymenko, L. H. Dudchenko, T. YU. Hrabova [ta inshi] - Fitoterapiya. Chasopys – №2 – K.: 2014 – S. 34 – 39.</p><p class="aCxSpMiddle">5. Ranozazhyvlyayushchaya aktyvnostʹ mazy, soderzhashchey éfyrnoe maslo travy tysyachelystnyka poymennoho / S. D. Trzhetsynskyy, V. Y. Mozulʹ, H. A. Zhernov [y dr.] – Naukovo - praktychnyy zhurnal – Zaporizhzhya : Aktualʹni pytannya farmatsevtychnoyi i medychnoyi nauky ta praktyky, № 2 (15) – 2014. – S. 50 – 53.</p><p class="aCxSpMiddle">6. Doklinichni doslidzhennya likarsʹkykh zasobiv: Metodychni rekomendatsiyi / Za red. chl.-kor. AMN Ukrayiny O. V. Stefanova. – K.: Avitsena, 2001. – 528 s.</p><p class="aCxSpMiddle">7. Teoryya y praktyka mestnoho lechenyya hnoynykh ran (problemy lekarstvennoy terapyy) / Pod red. B. M. Datsenko. – K.: Zdorov'ya, 1995. – 383 s.</p><p class="aCxSpMiddle">8. Rany y ranevaya ynfektsyya: Rukovodstvo dlya vrachey / Pod red. M. Y. Kuzyna, B. M. Kostyuchonok. – 2-e yzd. – M.: Medytsyna, 1990. – 592 s.</p><p class="aCxSpLast">9. Kamyshnykov V. S. Spravochnyk po klynyko - byokhymycheskoy laboratornoy dyahnostyke: V 2 t. – Mn.: Belarusʹ, 2000. – T. 1. – 495 s.</p>

Targeting Lon Protease Activity Promotes Growth Inhibition and Apoptosis in Acute Myeloid Leukemia Cell Lines

INTRODUCTIONAcute myeloid leukemia (AML) is a progressive malignant disorder of the myeloid lineage of hematopoietic cells, where overall three-year survival is below 20% for patients above 65 years (1). Therapy resistance or relapsed are the main causes of mortality among these patients. The functional changes that affect mitochondria may contribute to the development of cancer (2). The mitochondrial Lon, ubiquitously expressed in human cells and tissues, seems to be involved in protein quality control and stress response pathways in mitochondria. Several stress conditions induce an upregulation of Lon that may promote tumorigenesis by inhibiting apoptosis and promoting cell proliferation, transformation and angiogenesis (3). The role of Lon in AML has not been described yet. Our study aims to investigate if the activity of the protease may be linked to AML leukemogenesis and drug resistance. In particular we tested Lon protease activity inhibitors to clarify a new pathogenetic mechanism in controlling drug responsiveness in this haematological disorder.METHODSAML cell lines Kasumi-1 (acute myeloblastic leukemia), MOLM-13 (acute monocytic leukemia) and HL-60 (acute promyelocytic leukemia) were used to perform the experiments. Lon expression was measured by RT-PCR and immunoblotting. Cell-growth was quantified by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Reactive oxygen species (ROS) were evaluated by Nitro Blue Tetrazolium (NBT) assay. For cell cycle analysis, cells were fixed with ethanol, resuspended in phosphate-buffered saline (PBS) containing 0.1mg/mL RNase A, NP40 0,15% and 100mg/mL of propidium iodide (PI) and analysed by using flow cytometry and Modfit software. The early and late apoptosis was assessed with Annexin V-FITC Apoptosis Detection kit. To measure the mitochondrial potential, JC-1 Mitochondrial Membrane Potential assay kit was used.RESULTSLon protease was expressed at high levels in AML cell lines compared with resting or activated peripheral blood mononuclear cells and monocytes, but less than other cancer cell lines such as K562 (human erythromyeloblastoid leukemia), HDLM-2 (Hodgkin lymphoma, nuclear sclerosing), SHSY5Y (neuroblastoma), MCF7 (breast carcinoma). Based on previous works (4), which show that 2-Cyano-3,12-dioxooleana-1,9-dien-28-oic acid (CDDO) and its derivates inhibit the Lon protease activity, we evaluated the effect of two inhibitors on cell growth and the induction of apoptosis. The leukemic cell lines were treated with CDDO and CDDO-Me (CDDO methyl-ester) at different concentrations for 24, 48 and 72h. Both compounds inhibited the cell proliferation in a dose- and time-dependent manner and induced an increase of ROS. We observed a time-dependent increase in the percentage of Annexin V+ cells after treatment with CDDO (1,25µM) and CDDO-Me (0,25µM). The induction of apoptosis after CDDO exposure was important only at 72h, while we observed a percentage of apoptosis over 60% starting from 24h of CDDO-Me treatment. Interestingly, CDDO induced a time-dependent arrest in G2/M phase in MOLM-13 cells and in G0/G1 phase in Kasumi-1 and HL-60 cells. Instead the CDDO-Me induced an alteration of all cell cycle phases, probably for its cytotoxicity. Moreover CDDO-Me caused a higher decrease in mitochondrial potential (Dψm) than CDDO in all cell lines.CONCLUSIONIn conclusion, these findings show that Lon protease inhibitors promote growth inhibition and apoptosis and support the concept that mitochondrial Lon may represent a novel anticancer drug target.REFERENCES1. Burnett A, Wetzler M, Lowenberg B. Therapeutic advances in acute myeloid leukemia. J Clin Oncol. 2011, 29:487-494.2. Gogvadze V, Orrenius S, Zhivotovsky B. Mitochondria in cancer cells: what is so special about them? Trends Cell Biol. 2008, 18(4):165-73.3. Ngo JK, Pomatto LC, Davies KJ. Upregulation of the mitochondrial Lon Protease allows adaptation to acute oxidative stress but dysregulation is associated with chronic stress, disease, and aging. Redox Biol. 2013, 1(1):258-64.4. Bernstein SH, Venkatesh S, Li M, Lee J, Lu B, Hilchey SP, Morse KM, Metcalfe HM, Skalska J, Andreeff M, Brookes PS, Suzuki CK. The mitochondrial ATP-dependent Lon protease: a novel target in lymphoma death mediated by the synthetic triterpenoid CDDO and its derivatives. Blood. 2012, 119(14):3321-9. DisclosuresMartinelli:Novartis: Consultancy, Speakers Bureau; BMS: Consultancy, Speakers Bureau; Pfizer: Consultancy; ARIAD: Consultancy.

Aberrant Microtubule Organization and Wiskott-Aldrich Syndrome-like Defects in Platelets and Megakaryocytes of Profilin1-Deficient Mice

Wiskott-Aldrich syndrome (WAS) is a rare, X-chromosomal recessive disorder which is caused by mutations in the WAS gene and characterized by eczema, immunodeficiency and microthrombocytopenia (Thrasher and Burns, Nat Rev Immunol 2010). Interestingly, WAS protein (WASp)-deficient mice have normal-sized platelets and thus the molecular link between WAS mutations and its central hallmark microthrombocytopenia remains elusive.Profilin1 (Pfn1) is a key actin-regulating protein that, besides actin, interacts with phosphoinositides and multiple proline-rich proteins including the WAS protein (WASp)/WASp-interacting protein (WIP) complex (Witke, Trends Cell Biol 2004; Ramesh et al., Proc Natl Acad Sci USA, 1997). Interestingly, similar to WAS patients, mice with Pfn1-null megakaryocytes/platelets suffered from microthrombocytopenia. We identified accelerated platelet clearance by macrophages and pre-mature platelet release into the bone marrow compartment as the major cause of the reduced platelet count in Pfn1-deficient mice.Both, platelets from Pfn1-null mice and WAS patients contained abnormally organized and hyperstable microtubules. We next tested, if increased microtubule stability could account for the reduced size of Pfn1-deficient platelets. Treatment of control platelets with microtubule stabilizing toxins, such as the histone-deacetylase inhibitor trichostatin A (TSA) or taxol resulted in a decreased platelet size. This finding indicates that increased microtubule stability could account for the reduced platelet size in Pfn1-deficient mice but also in WAS patients. Based on these results we speculate that WASp might modulate Pfn1 function and dysregulation of this interaction leads to increased stability and altered organization of microtubules. In support of this, the subcellular localization of Pfn1 was altered in platelets of three WAS patients.Together, these results reveal an unexpected function of Pfn1 as a regulator of microtubule organization and point to a previously unrecognized mechanism underlying the platelet formation defect in WAS patients. DisclosuresNo relevant conflicts of interest to declare.

Abstract 1154: Age of type I collagen is critical for regulation of HT-1080 cell proliferation in 3D matrices

Abstract The tumor microenvironnement consists of cells, soluble factors, signaling molecules, extracellular matrix (EM), and mechanical cues that can promote neoplastic transformation, support tumor growth and invasion, and foster therapeutic resistance. In addition to provide a physical barrier against invasion, type I collagen, the most abundant protein in EM, is also known to play a role in tumor cell proliferation (1). DeClerck et al. (2) has reported a direct inhibitory effect of fibrillar type I collagen on tumor cell proliferation when compared to the monomeric one. Type I collagen is a life-long protein (half-life 15 yrs) susceptible to undergo non enzymatic post-translational modifications such as production of Advanced Glycation End Products (AGEs). These modifications are able to influence the behavior of tumor cells. Here, we investigated whether regulation of HT-1080 cell proliferation is a consequence of type I collagen aging in terms of fibrillar state of the protein (2,3), or of the level of AGEs, also known to stimulate cell proliferation (4). Rat tail type I collagen were prepared from adult (2 months) and old (2 years) animals. A significant increase in AGEs has been observed in collagen extracted from old rats, compared to the adult. Under 3D growth condition, HT-1080 cells proliferate rapidly in old type I collagen relative to the adult one. This effect was not observed in 2D coating culture. The low rate of proliferation in adult collagen is accompanied by a downregulation of ERK1/2 activation, and an upregulation of p21, the inhibitor of cell cycle progression. This age-dependent cell proliferation regulatory effect does not involve α2β1 integrins. Neither Pi3K/Akt nor JNK pathways were involved in this process. Accumulating evidence suggest that Discoidin Domain Receptor 2 (DDR2) is a Receptor Tyrosine Kinase (RTK) with the unique ability among RTKs to respond to fibrillar collagen (5). To determine whether a similar response is elicited by DDR2 in both collagens, DDR2 was immunoprecipitated from cell lysates, and phosphorylated DDR2 was detected by immunoblotting. High level of DDR2 phosphorylation was observed after 3 days culture in adult collagen compared to the old one, whereas DDR2 expression remains unchanged in the presence of both collagens. Moreover, the DDR2 kinase function inhibitor, nilotinib, restored cell proliferation in adult collagen to a level similar to that observed in the old one. Taken together, these data establish a role for DDR2 in critical events during restriction of tumor cell proliferation induced by adult type I collagen.

AB0179 Hypermethylation of HDAC6 Promoter in Systemic Lupus Erythematosus

BackgroundGenes and environmental factors are involved in the pathogenesis of systemic lupus erythematosus (SLE). DNA methylation and histone modification affect the binding of transcription factors, and alter the expression of...

Biophysical Underpinnings of the Repeat Length Dependence of Polyglutamine Amyloid Formation

There are now 10 expanded CAG repeat diseases in which both disease risk and age of onset are strongly dependent on the repeat length of the polyglutamine (polyQ) sequence in the disease protein. Large, polyQ-rich inclusions in patient brains and in cell and animal models are consistent with the involvement of polyQ aggregation in the disease mechanism. This possibility is reinforced by studies showing strong repeat length dependence to the aggregation process, qualitatively mirroring the repeat length dependence of disease risk. Our understanding of the underlying biophysical principles that mediate the repeat length dependence of aggregation, however, is far from complete. A previous study of simple polyQ peptides showed that N*, the size of the critical nucleus that controls onset of aggregation, decreases from unfavorable tetramer to favorable monomer over the range Q23 to Q26. These data, however, do not explain why, for all peptides exhibiting N* ∼ 1, spontaneous aggregation rates continue to increase with increasing repeat length. Here we describe a novel kinetics analyses that maps out the nonlinear dependence with repeat length of a nucleation efficiency term that is likely related to aspects of nucleus structure. This trend accounts for why nucleus size increases to tetrameric at repeat lengths of Q23 or below. Intriguingly, both aggregation and age of onset trend with repeat length in similar ways, exhibiting large changes per added Gln at low repeat lengths and small changes per added Gln at relatively long repeat lengths. Fibril stability also increases with repeat length in a nonlinear fashion.